Patient-derived tumoroids and proteomic signatures: tools for early drug discovery.

Onco-virotherapy is an emergent treatment for cancer based on viral vectors. The therapeutic activity is based on two different mechanisms including tumor-specific oncolysis and immunostimulatory properties. In this study, we evaluated onco-virotherapy in vitro responses on immunocompetent non-small cell lung cancer (NSCLC) patient-derived tumoroids (PDTs) and healthy organoids. PDTs are accurate tools to predict patient's clinical responses at the in vitro stage. We showed that onco-virotherapy could exert specific antitumoral effects by producing a higher number of viral particles in PDTs than in healthy organoids. In the present work, we used multiplex protein screening, based on proximity extension assay to highlight different response profiles. Our results pointed to the increase of proteins implied in T cell activation, such as IFN-γ following onco-virotherapy treatment. Based on our observation, oncolytic viruses-based therapy responders are dependent on several factors: a high PD-L1 expression, which is a biomarker of greater immune response under immunotherapies, and the number of viral particles present in tumor tissue, which is dependent to the metabolic state of tumoral cells. Herein, we highlight the use of PDTs as an alternative in vitro model to assess patient-specific responses to onco-virotherapy at the early stage of the preclinical phases.

Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors.

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

CXCR4-Specific Nanobodies as Potential Therapeutics for WHIM syndrome.

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement.

Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential 'on-off' switch of pDC activity with therapeutic potential.

Interaction of chemokine receptor CXCR4 in monomeric and dimeric state with its endogenous ligand CXCL12: coarse-grained simulations identify differences.

Despite the recent resolutions of the crystal structure of the chemokine receptor CXCR4 in complex with small antagonists or viral chemokine, a description at the molecular level of the interactions between the full-length CXCR4 and its endogenous ligand, the chemokine CXCL12, in relationship with the receptor recognition and activation, is not yet completely elucidated. Moreover, since CXCR4 is able to form dimers, the question of whether the CXCR4-CXCL12 complex has a 1:1 or 2:1 preferential stoichiometry is still an open question. We present here results of coarse-grained protein-protein docking and molecular dynamics simulations of CXCL12 in association with CXCR4 in monomeric and dimeric states. Our proposed models for the 1:1 and 2:1 CXCR4-CXCL12 quaternary structures are consistent with recognition and activation motifs of both partners provided by the available site-directed mutagenesis data. Notably, we observed that in the 2:1 complex, the chemokine N-terminus makes more steady contacts with the receptor residues critical for binding and activation than in the 1:1 structure, suggesting that the 2:1 stoichiometry would favor the receptor signaling activity with respect to the 1:1 association.

The CXCL12/CXCR4 Signaling Pathway: A New Susceptibility Factor in Human Papillomavirus Pathogenesis.

The productive human papillomavirus (HPV) life cycle is tightly linked to the differentiation and cycling of keratinocytes. Deregulation of these processes and stimulation of cell proliferation by the action of viral oncoproteins and host cell factors underlies HPV-mediated carcinogenesis. Severe HPV infections characterize the wart, hypogammaglobulinemia, infection, and myelokathexis (WHIM) immunodeficiency syndrome, which is caused by gain-of-function mutations in the CXCR4 receptor for the CXCL12 chemokine, one of which is CXCR41013. We investigated whether CXCR41013 interferes in the HPV18 life cycle in epithelial organotypic cultures. Expression of CXCR41013 promoted stabilization of HPV oncoproteins, thus disturbing cell cycle progression and proliferation at the expense of the ordered expression of the viral genes required for virus production. Conversely, blocking CXCR41013 function restored virus production and limited HPV-induced carcinogenesis. Thus, CXCR4 and its potential activation by genetic alterations in the course of the carcinogenic process can be considered as an important host factor for HPV carcinogenesis.

Integration of New Information with Active Memory Accounts for Retrograde Amnesia: A Challenge to the Consolidation/Reconsolidation Hypothesis?

Active (new and reactivated) memories are considered to be labile and sensitive to treatments disrupting the time-dependent consolidation/reconsolidation processes required for their stabilization. Active memories also allow the integration of new information for updating memories. Here, we investigate the possibility that, when active, the internal state provided by amnesic treatments is represented and integrated within the initial memory and that amnesia results from the absence of this state at testing. We showed in rats that the amnesia resulting from systemic, intracerebroventricular and intrahippocampal injections of the protein synthesis inhibitor cycloheximide, administered after inhibitory avoidance training or reactivation, can be reversed by a reminder, including re-administration of the same drug. Similar results were obtained with lithium chloride (LiCl), which does not affect protein synthesis, when delivered systemically after training or reactivation. However, LiCl can induce memory given that a conditioned taste aversion was obtained for a novel taste, presented just before conditioning or reactivation. These results indicate that memories can be established and maintained without de novo protein synthesis and that experimental amnesia may not result from a disruption of memory consolidation/reconsolidation. The findings more likely support the integration hypothesis: posttraining/postreactivation treatments induce an internal state, which becomes encoded with the memory, and should be present at the time of testing to ensure a successful retrieval. This integration concept includes most of the previous explanations of memory recovery after retrograde amnesia and critically challenges the traditional memory consolidation/reconsolidation hypothesis, providing a more dynamic and flexible view of memory. SIGNIFICANCE STATEMENT: This study provides evidence challenging the traditional consolidation/reconsolidation hypotheses that have dominated the literature over the past 50 years. Based on amnesia studies, that hypothesis states that active (i.e., new and reactivated) memories are similarly labile and (re)established in a time-dependent manner within the brain through processes that require de novo protein synthesis. Our data show that new/reactivated memories can be formed without protein synthesis and that amnesia can be induced by drugs that do not affect protein synthesis. We propose that amnesia results from memory integration of the internal state produced by the drug that is subsequently necessary for retrieval of the memory. This interpretation gives a dynamic view of memory, rapidly stored and easily updated when active.