The Role of ATRA, Natural Ligand of Retinoic Acid Receptors, on EMT-Related Proteins in Breast Cancer: Minireview

The knowledge of the structure, function, and abundance of specific proteins related to the EMT process is essential for developing effective diagnostic approaches to cancer with the perspective of diagnosis and therapy of malignancies. The success of all-trans retinoic acid (ATRA) differentiation therapy in acute promyelocytic leukemia has stimulated studies in the treatment of other tumors with ATRA. This review will discuss the impact of ATRA use, emphasizing epithelial-mesenchymal transition (EMT) proteins in breast cancer, of which metastasis and recurrence are major causes of death.

Novel Methodologies for the Synthesis of Multivalent Glycoconjugates

<p>Glycoconjugates, such as glycolipids and glycoproteins, play an important role in health and disease. The synthesis and biological evaluation of these glycoconjugates allows for the development of novel carbohydrate-based therapeutics and analytical tools. Traditionally, the conjugation of glycans to other substrates required the installment of an anomeric linker during the total synthesis of the glycan, however, this strategy does not allow for the conjugation of naturally isolated glycans. To address this concern, glycan conjugation methodologies without the need for protecting groups have been developed, including the use of oxyamine conjugation methodologies. In particular, the synthesis of a variety of novel bi-functional oxyamine linkers enabled the rapid assembly of various types of glycoconjugates, including fluorescent- and biotinylated-glycans, glycoproteins and multivalent glycodendrons. The multivalent presentation of glycans to cell surface lectins is often required to induce a measurable biological response. This multivalent binding can be achieved by the presentation of glycans on dendrons as these ‘glycodendrons’ have increased affinity for their corresponding lectin compared to monovalent glycans. Moreover, these glycodendrons have several advantages, including high synthetic control, low cytotoxicity and in addition can be derivatised with molecular probes of choice, which can aid in the biological evaluation of these glycoconjugates. Accordingly, novel biotinylated and fluorescent dendrons were synthesised from a highly convergent second generation dendron core scaffold. These functionalised dendrons then allow for the nonavalent conjugation of carbohydrates, such as Lewis antigens, for their biological evaluation in the selective targeting of lectins. Lewis antigens play an important role in host cell recognition, but these glycans are also involved in disease, such as in cancer metastasis and HIV-infection. The synthesis of Lewis antigens allows for the biological evaluation of these glycans, and moreover, could be employed in the development of novel glycan-based therapeutics and analytical tools. Accordingly, a novel high-yielding and efficient synthesis of a crystalline trisaccharide building block is presented, which can then be utilised in the synthesis of most Type-2 Lewis antigens. In particular, the global deprotection of the crystalline material gave the LewisX glycan antigen, a natural ligand for the C-Type lectin DC-SIGN on dendritic cells and macrophages. Finally, the rapid assembling of complex multivalent glycodendrons is discussed by conjugating the glycan antigens to the functionalised multivalent dendrons through the use of the bi-functional oxyamine linker methodology. In particular, the synthesis of a fluorescent LewisX glycodendron is presented, and to demonstrate a potential biological application of this methodology, the fluorescent LewisX glycodendron is evaluated as a flow cytometry marker for the C-type lectin DC-SIGN on human macrophages.</p>

Novel Methodologies for the Synthesis of Multivalent Glycoconjugates

<p>Glycoconjugates, such as glycolipids and glycoproteins, play an important role in health and disease. The synthesis and biological evaluation of these glycoconjugates allows for the development of novel carbohydrate-based therapeutics and analytical tools. Traditionally, the conjugation of glycans to other substrates required the installment of an anomeric linker during the total synthesis of the glycan, however, this strategy does not allow for the conjugation of naturally isolated glycans. To address this concern, glycan conjugation methodologies without the need for protecting groups have been developed, including the use of oxyamine conjugation methodologies. In particular, the synthesis of a variety of novel bi-functional oxyamine linkers enabled the rapid assembly of various types of glycoconjugates, including fluorescent- and biotinylated-glycans, glycoproteins and multivalent glycodendrons. The multivalent presentation of glycans to cell surface lectins is often required to induce a measurable biological response. This multivalent binding can be achieved by the presentation of glycans on dendrons as these ‘glycodendrons’ have increased affinity for their corresponding lectin compared to monovalent glycans. Moreover, these glycodendrons have several advantages, including high synthetic control, low cytotoxicity and in addition can be derivatised with molecular probes of choice, which can aid in the biological evaluation of these glycoconjugates. Accordingly, novel biotinylated and fluorescent dendrons were synthesised from a highly convergent second generation dendron core scaffold. These functionalised dendrons then allow for the nonavalent conjugation of carbohydrates, such as Lewis antigens, for their biological evaluation in the selective targeting of lectins. Lewis antigens play an important role in host cell recognition, but these glycans are also involved in disease, such as in cancer metastasis and HIV-infection. The synthesis of Lewis antigens allows for the biological evaluation of these glycans, and moreover, could be employed in the development of novel glycan-based therapeutics and analytical tools. Accordingly, a novel high-yielding and efficient synthesis of a crystalline trisaccharide building block is presented, which can then be utilised in the synthesis of most Type-2 Lewis antigens. In particular, the global deprotection of the crystalline material gave the LewisX glycan antigen, a natural ligand for the C-Type lectin DC-SIGN on dendritic cells and macrophages. Finally, the rapid assembling of complex multivalent glycodendrons is discussed by conjugating the glycan antigens to the functionalised multivalent dendrons through the use of the bi-functional oxyamine linker methodology. In particular, the synthesis of a fluorescent LewisX glycodendron is presented, and to demonstrate a potential biological application of this methodology, the fluorescent LewisX glycodendron is evaluated as a flow cytometry marker for the C-type lectin DC-SIGN on human macrophages.</p>

Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts

Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.

Novel Galectin-3 Roles in Neurogenesis, Inflammation and Neurological Diseases

Galectin-3 (Gal-3) is an evolutionarily conserved and multifunctional protein that drives inflammation in disease. Gal-3′s role in the central nervous system has been less studied than in the immune system. However, recent studies show it exacerbates Alzheimer’s disease and is upregulated in a large variety of brain injuries, while loss of Gal-3 function can diminish symptoms of neurodegenerative diseases such as Alzheimer’s. Several novel molecular pathways for Gal-3 were recently uncovered. It is a natural ligand for TREM2 (triggering receptor expressed on myeloid cells), TLR4 (Toll-like receptor 4), and IR (insulin receptor). Gal-3 regulates a number of pathways including stimulation of bone morphogenetic protein (BMP) signaling and modulating Wnt signalling in a context-dependent manner. Gal-3 typically acts in pathology but is now known to affect subventricular zone (SVZ) neurogenesis and gliogenesis in the healthy brain. Despite its myriad interactors, Gal-3 has surprisingly specific and important functions in regulating SVZ neurogenesis in disease. Gal-1, a similar lectin often co-expressed with Gal-3, also has profound effects on brain pathology and adult neurogenesis. Remarkably, Gal-3′s carbohydrate recognition domain bears structural similarity to the SARS-CoV-2 virus spike protein necessary for cell entry. Gal-3 can be targeted pharmacologically and is a valid target for several diseases involving brain inflammation. The wealth of molecular pathways now known further suggest its modulation could be therapeutically useful.

Rational Chemical and Genetic Modifications Enhance Avidity and Function of CD70-Directed CAR-T-Cells for Myeloid Leukemia

Chimeric Antigen Receptor T cells (CAR-T) have changed the therapeutic landscape for lymphoid malignancies, but not yet in myeloid malignancies like acute myeloid leukemia (AML). The TNF-alpha family member CD70 has emerged as a promising surface target antigen in AML after high complete response rates (CR) were seen in a Phase 1 trial of the CD70 antibody cusatuzumab (Riether et al, Nature Medicine 2020). Disappointingly, phase II results with cusatuzumab found CR rates less one-half those seen in the phase 1 (Trudel ASCO 2020). Because CAR-T cells may recognize lower antigen densities than monoclonal antibodies, we sought to develop a better CAR-T strategy for targeting the CD70 antigen in AML. A recent effort to improve on the 'first' generation natural-ligand based CD70 CAR (full length-CD27 fused to CD3zeta) compared a variety of CARs, including the single-chain variable-fragment as the binding moiety to CD70, found that the original 'first' generation CAR was superior (Shaffer Blood 2011, Sauer Blood 2021). Interestingly, a second generation natural-ligand based CAR that included the 4-1BB costimulatory domain was thought to be superior to first-generation (zeta-only) CARs with the same binder (Wang Clinical Cancer Research 2016). We first confirmed that the ligand-based 4-1BB (Native) CAR, had activity against multiple AML targets in standard CAR-T assays including activation, cytolysis, and demonstrated activity in a NOD-SCID IL2R γnull (NSG) Molm13 mouse model of AML. However, these models were not curative, even in combination with azacitidine, which we confirmed mediated increase CD70 expression on the AML cells. We hypothesized that surface cleavage of CD27, which is the natural ligand of CD70, attenuated the function of the Native CARs; we confirmed this hypothesis by measuring soluble CD27 in CAR T cells co-cultured with AML targets. To abrogate surface cleavage of ligand-based CARs, we generated and tested a panel of rationally designed, novel hinge CAR variants ('truncated', 'deleted', 'flexible', and 'CD8hinge&TM', Figure 1A). We found that the CD8hinge&TM variant had improved cytolysis against AML targets in vitro as well as higher binding avidity as measured by acoustic force microscopy (Figure 1B). Furthermore, there was no detectable soluble CD27 after co-culture with AML targets, suggesting successful abrogation of hinge cleavage. When all the CAR variants were compared in vivo to the Native CAR, CD8hinge&TM CARs mediated improved tumor control, had higher CAR expansion in blood and bone marrow, a persistent central memory phenotype beyond 30 days, and mediated improved survival (Figure 1C). The effect of the CD8hinge&TM CARs was further enhanced in combination with azacitidine. We also found that tumor control (maximum flux) correlated most strongly with relative binding avidity of the hinge variants (R 2 0.906) compared to other measures of in vitro function such as IFNg production (R 2 0.548) or in vitro cytolysis (R 2 0.5982). Finally, we tested the Native and CD8hinge&TM CARs in patient-derived xenograft (PDX) models and confirmed that the Native CAR T cells did not control AML tumor and resulted in uniform lethality, whereas the CD8hinge&TM CAR T cells had superior in vivo expansion and were able to mediate AML eradication in mice. Our findings demonstrate that natural-ligand binding domains of CARs targeting CD70 in AML can be effective but require mechanisms to overcome surface cleavage. CD70-targeted CARs comprised of a fusion of truncated CD27 to a CD8 hinge and transmembrane domain have promise in patients with AML, with and without combination with azacitidine. Figure 1 Figure 1. Disclosures Wehrli: Novartis: Current equity holder in publicly-traded company; Nestle: Current equity holder in publicly-traded company; CSL Behring: Patents & Royalties. Frigault: Editas: Consultancy; Takeda: Consultancy; Iovance: Consultancy; Arcellx: Consultancy; Kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy. Maus: Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy.

Cancer immune therapy with PD-1-dependent CD137 co-stimulation provides localized tumour killing without systemic toxicity

Expression of the cell surface receptor CD137 has been shown to enhance anti-cancer T cell function via engagement with its natural ligand 4-1BBL. CD137 ligation with engineered ligands has emerged as a cancer immunotherapy strategy, yet clinical development of agonists has been hindered by either toxicity or limited efficacy. Here we show that a CD137/PD-1 bispecific antibody, IBI319, is able to overcome these limitations by coupling CD137 activation to PD-1-crosslinking. In CT26 and MC38 syngeneic mouse tumour models, IBI319 restricts T cell co-stimulation to PD-1-rich microenvironments, such as tumours and tumour-draining lymph nodes, hence systemic (liver) toxicity arising from generalised T cell activation is reduced. Besides limiting systemic T cell co-stimulation, the anti-PD-1 arm of IBI319 also exhibits checkpoint blockade functions, with an overall result of T and NK cell infiltration into tumours. Toxicology profiling in non-human primates shows that IBI319 is a well-tolerated molecule with IgG-like pharmacokinetic properties, thus a suitable candidate for further clinical development.

Targeting Nanomaterials to Head and Neck Cancer Cells Using A Fragment of the Shiga Toxin as a Potent Natural Ligand

Head and Neck Cancer (HNC) is the seventh most common cancer worldwide with a 5-year survival from diagnosis of 50%. Currently, HNC is diagnosed by a physical examination followed by an histological biopsy, with surgery being the primary treatment. Here, we propose the use of targeted nanotechnology in support of existing diagnostic and therapeutic tools to prevent recurrences of tumors with poorly defined or surgically inaccessible margins. We have designed an innocuous ligand-protein, based on the receptor-binding domain of the Shiga toxin (ShTxB), that specifically drives nanoparticles to HNC cells bearing the globotriaosylceramide receptor on their surfaces. Microscopy images show how, upon binding to the receptor, the ShTxB-coated nanoparticles cause the clustering of the globotriaosylceramide receptors, the protrusion of filopodia, and rippling of the membrane, ultimately allowing the penetration of the ShTxB nanoparticles directly into the cell cytoplasm, thus triggering a biomimetic cellular response indistinguishable from that triggered by the full-length Shiga toxin. This functionalization strategy is a clear example of how some toxin fragments can be used as natural biosensors for the detection of some localized cancers and to target nanomedicines to HNC lesions.

Hesperidin Interaction with HMG-CoA-Reductase Enzyme in Hypercholesterolemia: A Study in Silico

Dyslipidemia is a degenerative disease occurred with increased levels of fat and cholesterol levels in blood. One of the proteins used as anti-cholesterol is an HMG-CoA-Reductase. Hesperidin in orange peel can reduce cholesterol levels by interacting with HMG-CoA-Reductase. To prove this, an in silico method was used by using swissdock.ch (http://swissdock.ch/docking#). The receptor protein in dyslipidemia was obtained from the RCSB Protein Data Bank (https://www.rcsb.org) namely HMG-CoA-reductase receptor with code PDB: 1HW9. The natural ligand, hesperidin, was obtained from PubChem with code: 10621 (https://pubchem.ncbi.nlm.nih.gov/). Protein was prepared by omitting the natural ligand residues present in the protein. Ligand and protein preparations were used by the chimera 1.15. The result of this study indicated that the interaction of hesperidin with several amino acid recidues was predicted to provide inhibitory activity on HMG-CoA reductase as the protein target. Inhibition of HMG-CoA reductase will reduce mevalonate synthesis so that cholesterol levels will decrease. Keywords: hesperidin, HMG-CoA-Reductase, cholesterol, dyslhahipidemia