Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Identification and characterization of stromal-like cells with CD207+/low CD1a+/low phenotype derived from histiocytic lesions - a perspective in vitro model for drug testing.

BACKGROUND: Histiocytoses are rare disorders manifested by increased proliferation of pathogenic myeloid cells sharing histological features with macrophages or dendritic cells and accumulating in various organs, i.a., bone and skin. Pre-clinical in vitro models that could be used to determine molecular pathways of the disease are limited, hence research on histiocytoses is challenging. The current study compares cytophysiological features of progenitor, stromal-like cells derived from histiocytic lesions (sl-pHCs) of three pediatric patients with different histiocytoses types and outcomes. The characterized cells may find potential applications in drug testing. METHODS: Molecular phenotype of the cells, i.e. expression of CD1a and CD207 (langerin), was determined using flow cytometry. Cytogenetic analysis included GTG-banded metaphases and microarray (aCGH) evaluation. Furthermore, the morphology and ultrastructure of cells were evaluated using a confocal and scanning electron microscope. The microphotographs from the confocal imaging were used to reconstruct the mitochondrial network and its morphology. Basic cytophysiological parameters, such as viability, mitochondrial activity, and proliferation, were analyzed using multiple cellular assays, including Annexin V/7-AAD staining, mitopotential analysis, BrdU test, clonogenicity analysis, and distribution of cells within the cell cycle. Biomarkers potentially associated with histiocytoses progression were determined using RT-qPCR at mRNA, miRNA and lncRNA levels. Intracellular accumulation of histiocytosis-specific proteins was detected with Western blot. Cytotoxicyty and IC50 of vemurafenib and trametinib were determined with MTS assay. RESULTS: Obtained cellular models, i.e. RAB-1, HAN-1, and CHR-1, are heterogenic in terms of molecular phenotype and morphology. The cells express CD1a/CD207 markers characteristic for dendritic cells, but also show intracellular accumulation of markers characteristic for cells of mesenchymal origin, i.e. vimentin (VIM) and osteopontin (OPN). In subsequent cultures, cells remain viable and metabolically active, and the mitochondrial network is well developed, with some distinctive morphotypes noted in each cell line. Cell-specific transcriptome profile was noted, providing information on potential new biomarkers (non-coding RNAs) with diagnostic and prognostic features. The cells showed different sensitivity to vemurafenib and trametinib. CONCLUSION: Obtained and characterized cellular models of stromal-like cells derived from histiocytic lesions can be used for studies on histiocytosis biology and drug testing.

A novel peptide derived from the mannose binding lectin inhibits LPS-activated TLR4/NF-κB signaling and suppresses ocular inflammation.

Uveitis is a major cause of vision impairment worldwide. Current treatments have limited effectiveness but severe complications. Mannose binding lectin (MBL) is an important protein of the innate immune system that binds to TLR4 and suppresses LPS-induced inflammatory cytokine secretion. MBL-mediated inhibition of inflammation via the TLR4 pathway and MBL-derived peptides might be a potential therapeutics. In this study, we designed a novel MBL-derived peptide, WP-17, targeting TLR4. Bioinformatics analysis was conducted for the sequence, structure and biological properties of WP-17. The binding of WP-17 to THP-1 cells was analyzed using flow cytometry. Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by immunofluorescence-histochemical analysis. Effects of WP-17 were studied in vitro using LPS-stimulated THP-1 cells and in vivo in endotoxin-induced uveitis (EIU). Our results showed that WP-17 could bind to TLR4 expressed on macrophages, thus downregulating the expression levels of MyD88, IRAK-4, and TRAF-6, and inhibiting the downstream NF-kB signaling pathway and LPS-induced expression of TNF-α and IL-6 in THP-1 cells. Moreover, in EIU rats, intravitreal pretreatment with WP-17 demonstrated significant inhibitory effects on ocular inflammation, attenuating the clinical and histopathological manifestations of uveitis, reducing protein leakage and cell infiltration into the aqueous humor, and suppressing TNF-α and IL-6 production in ocular tissues. In summary, our study provides the first evidence of a novel MBL-derived peptide that suppressed activation of the NF-кB pathway by targeting TLR4. The peptide effectively inhibited rat uveitis and may be a promising candidate for the management of ocular inflammatory diseases.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

TRPV4 Activation Increases the Expression of CD207 (Langerin) of Monocyte-Derived Langerhans Cells without Affecting their Maturation.

Langerhans cells (LCs) are the sole professional antigen-presenting cell normally found in the human epidermal compartment. Research into their physiological role is hindered by the fact that they are invariably activated during isolation from the skin. To overcome this challenge, we turned to a monocyte-derived LC (moLC) model, which we characterized with RNA sequencing, and compared the transcriptome of moLCs with that of donor-matched immature dendritic cells. We found that moLCs express markers characteristic of LC2 cells as well as TRPV4. TRPV4 is especially important in the skin because it has been linked to the conservation of the skin barrier, immunological responses, as well as acute and chronic itch, but we know little about its function on LCs. Our results show that TRPV4 activation increased the expression of Langerin and led to increased intracellular calcium concentration in moLCs. Regarding the functionality of moLCs, we found that TRPV4 agonism had a mitigating effect on their inflammatory responses because it decreased their cytokine production and T-cell activating capability. Because TRPV4 has emerged as a potential therapeutic target in dermatological conditions, it is important to highlight LCs as, to our knowledge, a previously unreported target of these therapies.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

A population of dermal Langerin+ dendritic cells promote the inflammation in mouse model of atopic dermatitis.

Cutaneous dendritic cells (DCs) have been implicated in the pathogenesis of atopic dermatitis (AD). However, the specific role of different subsets of DCs has not been well defined. This study aimed to investigate the contributions of Langerhans cells (LCs), resident dermal Langerin+ DCs (r-Langerin+ dDCs), and newly infiltrated inflammatory dermal Langerin+ DCs (i-Langerin+ dDCs) in an AD mouse model induced by the topical application of MC903. The result showed that depletion of i-Langerin+ dDCs in DTR mice after multiple diphtheria toxin (DT) injection significantly reduced thymic stromal lymphopoietin (TSLP) production in lesions and skin inflammation alleviation. However, depletion of LCs or r-Langerin+ dDCs didn't resulted in significant changes in skin inflammation of DTA or single DT injection-treated DTR mice compared with the wild-type (WT) mice. DT-treated DTR-WT chimeric mice with the depletion of bone marrow (BM)-derived i-Langerin+ dDCs resulted in markedly decreased skin inflammation than controls, while PBS-treated chimeric mice (DTR-WT) with only the depletion of r-Langerin+ dDCs showed inflammation comparable to that in WT mice. Furthermore, TSLP contributed to the upregulation of Langerin expression in BM-derived DCs and promoted the maturation of Langerin+ DCs. In summary, the present study demonstrated that the newly infiltrated inflammatory dermal Langerin+ DCs were essential for AD development and local TSLP production, and TSLP further promoted the production of BM-derived i-Langerin+ dDCs, which might maintain AD inflammation.

Mannobioside biomimetics that trigger DC-SIGN binding selectivity.

Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Mannose in vaccine delivery.

Adjuvants and vaccine delivery systems are used widely to improve the efficacy of vaccines. Their primary roles are to protect antigen from degradation and allow its delivery and uptake by antigen presenting cells (APCs). Carbohydrates, including various structures/forms of mannose, have been broadly utilized to target carbohydrate binding receptors on APCs. This review summarizes basic functions of the immune system, focusing on the role of mannose receptors in antigen recognition by APCs. The most popular strategies to produce mannosylated vaccines via conjugation and formulation are presented. The efficacy of mannosylated vaccines is discussed in detail, taking into consideration factors, such as valency and number of mannose in mannose ligands, mannose density, length of spacers, special arrangement of mannose ligands, and routes of administration of mannosylated vaccines. The advantages and disadvantages of mannosylation strategy and future directions in the development of mannosylated vaccines are also debated.

Identification of the Allosteric Binding Site for Thiazolopyrimidine on the C-Type Lectin Langerin.

Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.

Permissive aggregative group formation favors coexistence between cooperators and defectors in yeast.

In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, enriching cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a "green beard gene". Because of the heterophilic nature of the Flo1p bond however, we hypothesize that kin recognition is permissive and depends on the relative stability of the FLO1+/flo1- versus FLO1+/FLO1+ detachment force F. We combine single-cell measurements of adhesion, individual cell-based simulations of cluster formation, and in vitro flocculation to study the impact of relative bond stability on the evolutionary stability of cooperation. We identify a trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition at the expense of cooperative benefits. We show that the fitness of FLO1 cooperators decreases as their frequency in the population increases, arising from the observed permissive character (F+- = 0.5 F++) of the Flo1p bond. Considering the costs associated with FLO1 expression, this asymmetric selection often results in a stable coexistence between cooperators and defectors.

Monitoring Circulating CD207+CD1a+ Cells in Langerhans Cell Histiocytosis and Clinical Implications.

Langerhans cell histiocytosis (LCH) is a disorder characterized by an abnormal accumulation of CD207+ and CD1a+ cells in almost any tissue. Currently, there is a lack of prognostic markers to follow up patients and track disease reactivation or treatment response. Putative myeloid precursors CD207+ and CD1a+ cells were previously identified circulating in the blood. Therefore, we aim to develop a sensitive tracing method to monitor circulating CD207+ and CD1a+ cells in a drop of blood sample of patients with LCH. A total of 202 blood samples from patients with LCH and 23 controls were tested using flow cytometry. A standardized cellular score was defined by quantifying CD207+ and CD1a+ expression in monocytes and dendritic cells, based on CD11b, CD14, CD11c, and CD1c subpopulations, resulting in a unique value for each sample. The scoring system was validated by a receiver operating characteristic curve showing a reliable discriminatory capacity (area under the curve of 0.849) with a threshold value of 14, defining the presence of circulating CD207+ and CD1a+ cells. Interestingly, a fraction of patients with no evident clinical manifestation at the time of sampling also showed presence of these cells (29.6%). We also found a differential expression of CD207 and CD1a depending on the organ involvement, and a positive correlation between the cellular score and plasma inflammatory markers such as soluble CD40L, soluble IL-2Ra, and CXCL12. In conclusion, the analysis of circulating CD207 and CD1a cells in a small blood sample will allow setting a cellular score with minimal invasiveness, helping with prognostic accuracy, detecting early reactivation, and follow-up.

The mannose receptor ligands and the macrophage glycome.

A unique glycan-binding protein expressed in macrophages and some types of other immune cells is the mannose receptor (MR, CD206). It is an endocytic, transmembrane protein with multiple glycan-binding domains and different specificities in binding glycans. The mannose receptor is important as it has major roles in diverse biological processes, including regulation of circulating levels of reproductive hormones, homeostasis, innate immunity, and infections. These different functions involve the recognition of a wide range of glycans, and their nature is currently under intense study. But the mannose receptor is just one of many glycan-binding proteins expressed in macrophages, leading to an interest in the potential relationship between the macrophage glycome and how it may regulate cognate glycan-binding protein activities. This review focuses primarily on the mannose receptor and its carbohydrate ligands, as well as macrophages and their glycomes.

uPARAP/Endo180: a multifaceted protein of mesenchymal cells.

The urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) is already known to be a key collagen receptor involved in collagen internalization and degradation in mesenchymal cells and some macrophages. It is one of the four members of the mannose receptor family along with a macrophage mannose receptor (MMR), a phospholipase lipase receptor (PLA2R), and a dendritic receptor (DEC-205). As a clathrin-dependent endocytic receptor for collagen or large collagen fragments as well as through its association with urokinase (uPA) and its receptor (uPAR), uPARAP/Endo180 takes part in extracellular matrix (ECM) remodeling, cell chemotaxis and migration under physiological (tissue homeostasis and repair) and pathological (fibrosis, cancer) conditions. Recent advances that have shown an expanded contribution of this multifunctional protein across a broader range of biological processes, including vascular biology and innate immunity, are summarized in this paper. It has previously been demonstrated that uPARAP/Endo180 assists in lymphangiogenesis through its capacity to regulate the heterodimerization of vascular endothelial growth factor receptors (VEGFR-2 and VEGFR-3). Moreover, recent findings have demonstrated that it is also involved in the clearance of collectins and the regulation of the immune system, something which is currently being studied as a biomarker and a therapeutic target in a number of cancers.

LMAN1-MCFD2 complex is a cargo receptor for the ER-Golgi transport of α1-antitrypsin.

α1-antitrypsin (AAT) is a serine protease inhibitor synthesized in hepatocytes and protects the lung from damage by neutrophil elastase. AAT gene mutations result in AAT deficiency (AATD), which leads to lung and liver diseases. The AAT Z variant forms polymer within the endoplasmic reticulum (ER) of hepatocytes and results in reduction in AAT secretion and severe disease. Previous studies demonstrated a secretion defect of AAT in LMAN1 deficient cells, and mild decreases in AAT levels in male LMAN1 and MCFD2 deficient mice. LMAN1 is a transmembrane lectin that forms a complex with a small soluble protein MCFD2. The LMAN1-MCFD2 protein complex cycles between the ER and the Golgi. Here, we report that LMAN1 and MCFD2 knockout (KO) HepG2 and HEK293T cells display reduced AAT secretion and elevated intracellular AAT levels due to a delayed ER-to-Golgi transport of AAT. Secretion defects in KO cells were rescued by wild-type LMAN1 or MCFD2, but not by mutant proteins. Elimination of the second glycosylation site of AAT abolished LMAN1 dependent secretion. Co-immunoprecipitation experiment in MCFD2 KO cells suggested that AAT interaction with LMAN1 is independent of MCFD2. Furthermore, our results suggest that secretion of the Z variant, both monomers and polymers, is also LMAN1-dependent. Results provide direct evidence supporting that the LMAN1-MCFD2 complex is a cargo receptor for the ER-to-Golgi transport of AAT and that interactions of LMAN1 with an N-glycan of AAT is critical for this process. These results have implications in production of recombinant AAT and in developing treatments for AATD patients.

BMPR1a Is Required for the Optimal TGFß1-Dependent CD207+ Langerhans Cell Differentiation and Limits Skin Inflammation through CD11c+ Cells.

The cytokine TGFß1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFß1‒BMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFß1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav+) hematopoietic cells revealed that BMPR1a is required for the efficient TGFß1-dependent generation of CD207+ LC-like cells from CD11c+ intermediates in vitro. Similarly, BMPR1a was required for the optimal induction of CD207 by preformed major histocompatibility complex II‒positive epidermal resident LC precursors in the steady state. BMPR1a expression is strongly upregulated in epidermal cells in psoriatic lesions, and BMPR1aΔCD11c mice showed a defect in the resolution phase of allergic and psoriatic skin inflammation. Moreover, whereas LCs from these mice expressed CD207, BMPR1a counteracted LC activation and migration from skin explant cultures. Therefore, TGFß1‒BMPR1a signaling seems to be required for the efficient induction of CD207 during LC differentiation in the steady state, and bone marrow‒derived lesional CD11c+ cells may limit established skin inflammation through enhanced BMPR1a signaling.

Specific Protein Antigen Delivery to Human Langerhans Cells in Intact Skin.

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.