Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells.

BACKGROUND: Despite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis and programmed cancer cell death are therapeutic targets to combine with immunotherapy to improve treatment success. In a recent study, we uncovered that a molecule called QAPHA ((E)-3-(5-((2-cyanoquinolin-4-yl)(methyl)amino)-2-methoxyphenyl)-N-hydroxyacrylamide) has a dual function as both a tubulin polymerization and HDAC inhibitors. Here, we investigate the impact of this novel dual inhibitor on the immune response to lung cancer. METHODS: To elucidate the mechanism of action of QAPHA, we conducted a chemical proteomics analysis. Using an in vivo mouse model of lung cancer (TC-1 tumor cells), we assessed the effects of QAPHA on tumor regression. Tumor infiltrating immune cells were characterized by flow cytometry. RESULTS: In this study, we first showed that QAPHA effectively inhibited histone deacetylase 6, leading to upregulation of HSP90, cytochrome C and caspases, as revealed by proteomic analysis. We confirmed that QAPHA induces immunogenic cell death (ICD) by expressing calreticulin at cell surface in vitro and demonstrated its efficacy as a vaccine in vivo. Remarkably, even at a low concentration (0.5 mg/kg), QAPHA achieved complete tumor regression in approximately 60% of mice treated intratumorally, establishing a long-lasting anticancer immune response. Additionally, QAPHA treatment promoted the infiltration of M1-polarized macrophages in treated mice, indicating the induction of a pro-inflammatory environment within the tumor. Very interestingly, our findings also revealed that QAPHA upregulated major histocompatibility complex class II (MHC-II) expression on TC-1 tumor cells both in vitro and in vivo, facilitating the recruitment of cytotoxic CD4+T cells (CD4+CTL) expressing CD4+, NKG2D+, CRTAM+, and Perforin+. Finally, we showed that tumor regression strongly correlates to MHC-II expression level on tumor cell and CD4+ CTL infiltrate. CONCLUSION: Collectively, our findings shed light on the discovery of a new multitarget inhibitor able to induce ICD and MHC-II upregulation in TC-1 tumor cell. These two processes participate in enhancing a specific CD4+ cytotoxic T cell-mediated antitumor response in vivo in our model of lung cancer. This breakthrough suggests the potential of QAPHA as a promising agent for cancer treatment.

In search of the cell biology for self- versus non-self- recognition.

Several of today's cancer treatments are based on the immune system's capacity to detect and destroy cells expressing neoantigens on major histocompatibility class-I molecules (MHC-I). Despite this, we still do not know the cell biology behind how antigenic peptide substrates (APSs) for the MHC-I pathway are produced. Indeed, there are few research fields with so many divergent views as the one concerning the source of APSs. This is quite remarkable considering their fundamental role in the immune systems' capacity to detect and destroy virus-infected or transformed cells. A better understanding of the processes generating APSs and how these are regulated will shed light on the evolution of self-recognition and provide new targets for therapeutic intervention. We discuss the search for the elusive source of MHC-I peptides and highlight the cell biology that is still missing to explain how they are synthesised and where they come from.

Major histocompatibility class I antigenic peptides derived from translation of pre-mRNAs generate immune tolerance.

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA.

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.

Design, synthesis and biological evaluation of quinoline-2-carbonitrile-based hydroxamic acids as dual tubulin polymerization and histone deacetylases inhibitors.

A series of quinoline and quinazoline analogs were designed and synthesized as new tubulin polymerization (TP) and histone deacetylases (HDAC) inhibitors. Compounds 12a and 12d showed the best cytotoxicity activities against a panel of human cancer cell lines with an averaged IC50 value of 0.6 and 0.7 nM, respectively. Furthermore, these lead compounds showed good activities against CA-4-resistant colon-carcinoma and multidrug-resistant leukemia cells. In addition, compounds 12a and 12d induced HT29 cell cycle arrest in the G2/M phase and produced caspase-induced apoptosis of HT29 cells through mitochondrial dysfunction. Also, 12a and 12d inhibited HDAC8, 6, and 11 activities. Furthermore, lead compound 12a exhibited higher metabolic stability than isoCA-4 and was highly potent in suppressing tumor growth in the fibrosarcoma MCA205 tumor model. Collectively, these studies suggest that 12a represents a new dual inhibitor of TP and HDAC activities, which makes it a suitable candidate for further investigations in clinical development.

Biological Investigation of a Water-Soluble Isoginkgetin-Phosphate Analogue, Targeting the Spliceosome with In Vivo Antitumor Activity.

The first total synthesis of the natural product Isoginkgetin as well as four water-soluble Isoginkgetin-phosphate analogues is reported herein. Moreover, the full study of the IP2 phosphate analogue with respect to pharmacological properties (metabolic and plasmatic stabilities, pharmacokinetic, off-target, etc.) as well as in vitro and in vivo biological activities are disclosed herein.

mRNA translation from an antigen presentation perspective: A tribute to the works of Nilabh Shastri.

The field of mRNA translation has witnessed an impressive expansion in the last decade. The once standard model of translation initiation has undergone, and is still undergoing, a major overhaul, partly due to more recent technical advancements detailing, for example, initiation at non-AUG codons. However, some of the pioneering works in this area have come from immunology and more precisely from the field of antigen presentation to the major histocompatibility class I (MHC-I) pathway. Despite early innovative studies from the lab of Nilabh Shastri demonstrating alternative mRNA translation initiation as a source for MHC-I peptide substrates, the mRNA translation field did not include these into their models. It was not until the introduction of the ribo-sequence technique that the extent of non-canonical translation initiation became widely acknowledged. The detection of peptides on MHC-I molecules by CD8 + T cells is extremely sensitive, making this a superior model system for studying alternative mRNA translation initiation from specific mRNAs. In view of this, we give a brief history on alternative initiation from an immunology perspective and its fundamental role in allowing the immune system to distinguish self from non-self and at the same time pay tribute to the works of Nilabh Shastri.

PA28γ-20S proteasome is a proteolytic complex committed to degrade unfolded proteins.

PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.

Isoginkgetin derivative IP2 enhances the adaptive immune response against tumor antigens.

The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8+ T cell response against TAs. We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis.

Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex-independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3.

The success of CD8+ T cell-based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and the possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during a pioneer round of mRNA translation (pioneer translation products, or PTPs) are an important source of tumor specific peptides which correlates with the aberrant splicing and transcription events associated with oncogenesis. Here we show that up-regulation of PSME3 proteasome activator in cancer cells results in increased destruction of PTP-derived peptides in the nucleus thus enabling cancer cell to subvert immunosurveillance. These findings unveil a previously unexpected role for PSME3 in antigen processing and identify PSME3 as a druggable target to improve the efficacy of cancer immunotherapy.

Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides.

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.

A fluorine scan of a tubulin polymerization inhibitor isocombretastatin A-4: Design, synthesis, molecular modelling, and biological evaluation.

A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC50 values of 0.15-2.2 nM (3i) and 0.1-2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules organization showed that compound 3m induced G2/M phase arrest and, dramatically disrupted the microtubule network.

Exosome-driven transfer of tumor-associated Pioneer Translation Products (TA-PTPs) for the MHC class I cross-presentation pathway.

Cellular immune reactions against non-self-epitopes require activation of cytotoxic CD8+ T-cells via cross-presentation of MHC class I-restricted peptides by professional antigen presenting cells (pAPCs), with the consequent detection and elimination of cells expressing the same antigens via the endogenous (direct) pathway. The source of peptides for the endogenous pathway is constituted of alternative mRNA translation products; however, it is still unclear which source of peptides is used for cross-presentation. Furthermore, the presentation of non-canonical translation products, produced during a non-conventional translation event, on class I molecules of tumor cells has been reported but how these peptides are generated, presented to pAPCs, and their capacity to stimulate CD8+ T cells is still not known. Here, we report that pioneer translation peptides (PTPs) derived from intron or exon pre-mRNAs can serve as tumor-associated antigens (TA-PTPs) and are delivered from the producing tumor cells to pAPCs via exosomes where they are processed by the cytosolic pathway. Injection of TA-PTPs and tumor-derived exosomes efficiently induce CD8+ T-cell proliferation and prevent tumor growth in mice. Our results show that TA-PTPs represent an efficient source of antigenic peptides for CD8+ T cell activation and that full-length proteins are not required for cross-presentation. These findings can have interesting implications for generating tolerance and for designing vectors to generate vaccines.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

The source of MHC class I presented peptides and its implications.

The source of peptides that enter the major histocompatibility class I (MHCI) pathway has been intensively debated over the last two decades. The initial assumption that peptides are derived from degradation of full length proteins was challenged by a model in which alternative translation products are a source of peptides. This model has been tested and supported by scientific data. We now need new hypotheses on the physiological implications of different sources of peptides for the MHCI pathway. The aim of this overview is to give an up-to-date account of the source of antigenic peptide material for the MHCI pathway and to incorporate the more recent observations of alternative mRNA translation products into existing models of the direct and cross-presentation pathways.

Pioneer translation products as an alternative source for MHC-I antigenic peptides.

The notion that alternative peptide substrates can be processed and presented to the MHC class I pathway has opened for new aspects on how the immune system detects infected or damaged cells. Recent works show that antigenic peptides are derived from intron sequences in pre-mRNAs target for the nonsense-mediated degradation pathway. Introns are spliced out co-transcriptionally suggesting that such pioneer translation products (PTPs) are synthesized on the nascent RNAs in the nuclear compartment to ensure that the first peptides to emerge from an mRNA are destined for the class I pathway. This illustrates an independent translation event during mRNA maturation that give rise to specific peptide products with a specific function in the immune system. The characterization of the translation apparatus responsible for PTP synthesis will pave the way for understanding how PTP production is regulated in different tissues under different conditions and will help designing new vaccine strategies.

Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers.

The EBV-encoded EBNA1 was first discovered 40 years ago, approximately 10 years after the presence of EBV had been demonstrated in Burkitt's lymphoma cells. It took another 10 years before the functions of EBNA1 in maintaining the viral genome were revealed, and it has since been shown to be an essential viral factor expressed in all EBV-carrying cells. Apart from serving to maintain the viral episome and to control viral replication and gene expression, EBNA1 also harbours a cis-acting mechanism that allows virus-carrying host cells to evade the immune system. This relates to a particular glycine-alanine repeat (GAr) within EBNA1 that has the capacity to suppress antigen presentation to the major histocompatibility complex (MHC) class I pathway. We discuss the role of the GAr sequence at the level of mRNA translation initiation, rather than at the protein level, as at least part of the mechanism to avoid MHC presentation. Interfering with this mechanism has become the focus of the development of immune-based therapies against EBV-carrying cancers, and some lead compounds that affect translation of GAr-carrying mRNAs have been identified. In addition, we describe the EBV-encoded ZEBRA factor and the switch from the latent to the lytic cycle as an alternative virus-specific target for treating EBV-carrying cancers. Understanding the molecular mechanisms of how EBNA1 and ZEBRA interfere with cellular pathways not only opens new therapeutic approaches but continues to reveal new cell-biological insights on the interplay between host and virus. This review is a tale of discoveries relating to how EBNA1 and ZEBRA have emerged as targets for specific cancer therapies against EBV-carrying diseases, and serves as an illustration of how mRNA translation can play roles in future immune-based strategies to target viral disease.

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus.

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8(+) T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II-DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II-DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.