Transmembrane Glycoprotein CD44 Research Articles

Preparation and Characterization of Collagen-Hyaluronic Acid (Col-HA) Matrices: In Vitro Mimics of the Tumor Microenvironment.

Collagen-I (Col) and hyaluronic acid (HA) are two of the most important extracellular matrix (ECM) components. While collagen serves as the main structural feature of the ECM and is an important ligand for integrin-based adhesions, HA acts as a ligand for the transmembrane glycoprotein CD44. However, most studies on cancer invasion utilize 3D collagen matrices, thereby ignoring the importance of HA-CD44 interactions. Here, we describe the process of fabricating Col-HA gels with varying Col/HA ratios, which can be used to suitably mimic the tumor microenvironment.

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblastsionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

New Horizons in Metastatic Colorectal Cancer: Prognostic Role of CD44 Expression

The transmembrane glycoprotein CD44, the major hyaluronan (HA) receptor, has been proven to regulate cell growth, survival, differentiation, and migration. It is therefore widely considered to be involved in carcinogenesis. Its role as a new therapeutic target in solid tumors is under evaluation in clinical trials. The prognostic value remains controversial. Here, we aimed to investigate the correlation between CD44 expression and the clinicopathological features and survival in metastatic colorectal cancer (mCRC) patients. Data from 65 mCRC patients of the Medical Oncology Unit, University Hospital and University of Cagliari were retrospectively collected from 2008 to 2021. Immunohistochemical analysis was performed at the Pathology Division, University Hospital of Cagliari on 3 μm thick sections obtained from paraffin blocks. The intensity of immunohistochemical staining was subclassified into four groups: score 0 if negative or weak membrane staining in less than 10% of tumor cells; score 1+ if weak membrane staining in at least 10% of tumor cells or moderate membrane staining in less than 10% of tumor cells; score 2+ if moderate membrane staining in at least 10% of tumor cells or intensive membrane staining in less than 10% of tumor cells; score 3+ if intense membrane staining in at least 10% of tumor cells. Based on this score, we distinguished patients into low CD44 expression (score 0, 1+, 2+) and high CD44 expression (score 3+). Statistical analysis was performed with MedCalc (survival distribution: Kaplan-Meier; survival comparison: log-rank test; association between categorical variables: Fisher's exact test). Patients' median age was 66 years (range 49-85). Regarding CD44 expression, score was 0 in 18 patients, 1+ in 15 patients, 2+ in 18 patients, and 3+ in 14 patients. Median overall survival (mOS) was 28.1 months (95%CI: 21.3-101). CD44 overexpression (3+) was correlated with poor prognosis (p = 0.0011; HR = 0.2), with a mOS of 14.5 months (95%CI 11.7 to 35.9) versus 30.7 months (95%CI 27.8 to 101) in lower CD44 expression. Higher CD44 expression was associated with clinically poor prognostic features: age ≥ 70 years (p = 0.0166); inoperable disease (p = 0.0008); stage IV at diagnosis (p = 0.0241); BRAF mutated (p = 0.0111), high-grade tumor (p = 0.0084). CD44 markedly correlated with aggressive tumor behavior and contributed to the earlier progression of disease, thus suggesting its role as a novel prognostic marker and potential therapeutic target for mCRC patients.

CD44 Glycosylation as a Therapeutic Target in Oncology.

The interaction of non-kinase transmembrane glycoprotein CD44 with ligands including hyaluronic acid (HA) is closely related to the occurrence and development of tumors. Changes in CD44 glycosylation can regulate its binding to HA, Siglec-15, fibronectin, TM4SF5, PRG4, FGF2, collagen and podoplanin and activate or inhibit c-Src/STAT3/Twist1/Bmi1, PI3K/AKT/mTOR, ERK/NF-κB/NANOG and other signaling pathways, thereby having a profound impact on the tumor microenvironment and tumor cell fate. However, the glycosylation of CD44 is complex and largely unknown, and the current understanding of how CD44 glycosylation affects tumors is limited. These issues must be addressed before targeted CD44 glycosylation can be applied to treat human cancers.

CD38 a biomarker and therapeutic target in non-hematopoietic tumors.

The type II transmembrane glycoprotein CD38 has recently been implicated in regulating metabolism and the pathogenesis of multiple conditions, including aging, inflammation and cancer. CD38 is overexpressed in several tumor cells and microenvironment tumoral cells, associated to migration, angiogenesis, cell invasion and progression of the disease. Thus, CD38 has been used as a progression marker for different cancer types as well as in immunotherapy. This review focuses on describing the involvement of CD38 in various non-hematopoietic cancers.

Targeting CD38 in acute myeloid leukemia interferes with leukemia trafficking and induces phagocytosis

Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects.

Contribution of astroglial CD38 to opioid-mediated spinal antinociception in neuropathic and inflammatory pain models

Introduction: To determine the role of the transmembrane glycoprotein CD38, which is important for intracellular calcium homeostasis, in spinal opioid antinociception in chronic neuropathic and inflammatory pains, and to explore the cellular expression of CD38 in the spinal cord.

490P New horizons in metastatic colorectal cancer: Role of CD44 expression

The transmembrane glycoprotein CD44, the major hyaluronan receptor, is considered to be involved in carcinogenesis. Its role as new therapeutic molecular target in solid tumors is under evaluation in clinical trials. The prognostic value remains controversial. Here, we aimed to investigate the correlation between CD44 expression and the clinicopathological features and survival of metastatic colorectal cancer (mCRC) patients (pts).

Abstract 3116: Designing a model system for the study of CD44-Ezrin interactions in breast cancer progression and drug resistance

Abstract Triple-negative breast cancer (TNBC) has the highest risk of relapse and is thought to be the most aggressive subtype of BC. This subtype is known to express high levels of the cancer stem cell (CSC) marker CD44. The tumor-initiating properties of CSC's, as well as their resistance to chemotherapy, contribute to the difficulty in successfully treating TNBCs.CD44 is a transmembrane glycoprotein that mediates communication between cells and the extracellular matrix. In doing so, it acts as a central node in several cancer-related signaling pathways through interactions with binding partners such as Ezrin to promote tumor growth, invasion and survival. Furthermore, expression of CD44 is associated clinically with positive lymph node status, recurrence and poor overall survival in BC. We aimed to validate CD44's role in these cancer promoting properties and develop a genetically tractable and engraftable TNBC cell model to study CD44 in the context of interactions with the Ezrin adaptor protein. We first hypothesize that disrupting CD44 expression will attenuate the metastatic and drug resistant potential of TNBC cells. To address this, we utilized CRISPR-Cas9 gene editing to knockout (KO) CD44 expression in the MDA-MB-231 human TNBC cell line and then subjected CD44 KO cells, KO cells rescued with wild type CD44, or the parental MDA-MB-231 cells to a variety of in vitro assays to assess proliferative, migratory and drug resistant potential, as well as mouse lung metastatic seeding in vivo. We show that the transmembrane glycoprotein CD44 plays an important role in cancer cell proliferation, migration, and chemotherapeutic resistance in vitro as well as having an essential role in metastatic seeding in vivo in the MDA-MB-231 cell model of TNBC. Our results validate the utility of this CRISPR-Cas9 KO and rescue TNBC cell model for the study of CD44 involvement in drug resistance and metastasis. More importantly we have set the stage for further investigating CD44's role in the context of interactions with the Ezrin adaptor protein. Citation Format: Rayanna Jane Birtch, Peter Greer, Yan Gao, Victoria Hoskin. Designing a model system for the study of CD44-Ezrin interactions in breast cancer progression and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3116.

Single Cell Resolution Profiling Defines the Innate and Adaptive Immune Repertoires Modulated By Daratumumab and IMiDs Treatment in Multiple Myeloma (MM)

Background: The combination of immunomodulatory (IMiDs) drugs with monoclonal antibodies targeting the transmembrane glycoprotein CD38 induces deep and durable responses in MM patients. IMiDs mediated degradation of the transcription factors IKZF1/3 attenuates myeloma cells proliferation and releases IKZF1 transcriptional repression of IL-2 and type I/II interferon response genes promoting T, NK and NKT cells proliferation and activation. Similarly targeting the non-lineage restricted molecule CD38 with daratumumab also exerts direct and indirect tumoricidal effects. Mechanisms implicated in daratumumab-mediated killing include a direct apoptotic effect and activation of cytotoxic immune effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and complement dependent cytotoxicity (CDC). Therefore, the synergistic effects observed with daratumumab and IMiDs are postulated to derive from their co-modulation of the host adaptive and innate immunity. A systematic unsupervised interrogation of the bone marrow immune cells of daratumumab and IMiDs treated MM patients is key for the prediction of their clinical responses and the understanding of underlying mechanisms of resistance.Methods and Results: Bone marrow (BM) aspirates and matching peripheral blood were collected from 20 myeloma patients treated with daratumumab in combination with IMiDs (lenalidomide or pomalidomide) prior to initiation of therapy and at the time of disease response (n=10) or progression (n=10). Bone marrow mononuclear cells were isolated through Ficoll density gradients coupled with magnetic sorting of CD138pos and CD138neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics), a droplet-based method enabling high cell throughput and efficient single-cell capture. Sequencing was performed on Illumina NEXTseq platform in a paired-end format. Cell Ranger Single Cell Software Suite was used to perform sample de-multiplexing, barcode processing, and single-cell 3′ gene counting. Sequencing data from 30,074 CD138neg single cells (12,908 cells from responding (S) and 17,166 cells from progressing (R) patients) were kept for analysis by the Seurat R package and analyzed by principal component analysis (PCA) and clusteringand then visualized by t -distributed stochastic neighbour embedding (t -SNE) projection. Shown in Fig 1 a representative t -SNE and heatmap plot of 9 immune clusters typically identified in individual samples including terminal effector T cells (KLRG1hi, IL7Rlow), memory T cells (central memory TCM: SELLhi CCR7hi and effector memory TEM: SELLlow CCR7low), CD4 T cells, NK and NKT cells (NKG7, GNLY), B cells (MS4A1), monocytes (CD14, LYZ), macrophages (CD68) and dendritic cells (FCER1A, CST3). Transcriptome profiling of immune cells pre- and post-treatments in S and R patients revealed the following key findings: 1) T cells (CD8Apos and CD3pos) were largely senescent in myeloma patients lacking CD28 expression; 2) amongst checkpoint inhibitory genes LAG3 had the highest expression on activated T cells followed by PDCD1 and to a much lesser extent by HAVCR2 and TIGIT; 3) exposure to daratumumab and IMiDs resulted in the expansion of activated effector CD8 cyctotoxic and effector memory T cells and depletion of CD38pos T and B cells; 4) compared to resistant or progressing patients, responders were characterized by higher CD28 expression in T cells, a significantly larger clusters of central memory T cells (TCM: IL7Rpos, SELLpos, CCR7pos, CD27pos, KLRG1neg) and a M1 activated macrophage signature (IL1Bpos, CCL3pos). Of note no significant difference was noted in CD38 transcript amongst S and R patients. Single cell profiling of CD138pos cells will be updated at the meeting as well as further characterization of the immune repertoire and TCR clonality status of R and S patients.Conclusion: Anunbiased single cell profiling of bone marrow immune cells in MM patients exposed to Daratumumab and IMiDs demonstrated that LAG3 targeting and restoration of co-stimulatory genes expression (CD28) should be studied as means to reverse immunoparesis. Central memory T cells (TCM) expansion and M1 macrophage signature in responders to Daratumumab and IMiDs combinations represent potential biomarkers of response. [Display omitted] DisclosuresNeri:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Jimenez-Zepeda:Janssen: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Takeda: Honoraria. Boise:Eli Lilly and Company: Research Funding; Abbvie: Consultancy. Thakurta:Celgene Corporation: Employment, Equity Ownership. Bahlis:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The Immunomodulatory Effect and Clinical Efficacy of Daratumumab in a Patient With Cold Agglutinin Disease

Daratumumab is a monoclonal antibody directed against the transmembrane glycoprotein CD38 expressed on plasma cells and lymphoplasmocytes, with a proven efficacy in multiple myeloma. Here we show its clinical efficacy in a patient with cold agglutinin disease (CAD) relapsed after multiple lines of therapy. CAD is caused by cold reactive autoantibodies that induce complement mediated hemolysis and peripheral circulatory symptoms. The disease is also characterized by the presence of monoclonal IgM gammopathy and of a lymphoid bone marrow infiltration that benefits from B-cell targeting therapies (i.e., rituximab) but also from plasma cell directed therapies, such as proteasome inhibitors. In the patient described, we also show that daratumumab therapy influenced the dynamics of several immunoregulatory cytokine levels (IL-6, IL-10, IL-17, IFN-γ, TNF-α, TGF-β) indicating an immunomodulatory effect of the drug beyond plasma cell depletion. In addition, we provide a literature review on the use of daratumumab in autoimmune conditions, including multi-treated and refractory patients with autoimmune hemolytic anemia (both CAD and warm forms), Evans syndrome (association of autoimmune hemolytic anemia and immune thrombocytopenia) and non-hematologic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.

BIOMECHANICS AND FUNCTIONAL REGULATIONS OF CD44-LIGAND INTERACTIONS

As a widely expressed cellular adhesion molecule, type I transmembrane glycoprotein CD44 is crucial in cell proliferation, differentiation, migration, angiogenesis and other biological processes to induce intracellular signal transduction and regulate tissue homeostasis. Especially, cell adhesion dynamics mediated by CD44-selectin and CD44-hyaluronic acid (HA) interactions play key roles in classic inflammatory cascade, tumor metastasis, or tissue-specific liver immunity. This review discussed the progresses and remaining issues of CD44 selectin and CD44-HA interactions in various aspects of cellular adhesion dynamics, two- and three-dimensional molecular reaction kinetics, atomic microstructural features, and intracellular signal transduction pathways. Nowadays, the importance of mechanical and physical factors to biological activities has been gradually accepted by scientific community. New concepts such as mechanomedicine, mechanoimmunology and mechanomics have been put forward one after another. Under physiological or pathological conditions, cell adhesion mediated by CD44-ligand interactions are regulated by in vivo mechanical and physical cues such as blood shear or tissue stiffness, but their regulatory mechanisms are still unclear. From that on, future perspectives related to CD44-ligand interaction were also proposed in this review as follows: how mechanical and physical factors regulate cellular adhesion dynamics and intrinsic mechanism mediated by CD44-ligand interactions; what the mechanical regulation features of molecular reaction kinetics of CD44-ligand interactions and corresponding structural bases are; and how the atomic-level microstructures of CD44-ligand binding evolve dynamically under mechanical forces. This review provides clues for further understanding the biological functions and structure-function relationship of CD44-ligand interactions.

Development of a Hyaluronic Acid-Based Nanocarrier Incorporating Doxorubicin and Cisplatin as a pH-Sensitive and CD44-Targeted Anti-Breast Cancer Drug Delivery System.

Tumor-targeting nanomaterial-based chemotherapeutic drug delivery systems have been shown to represent an efficacious approach for the treatment of cancer because of their stability in blood circulation and predictable delivery patterns, enhanced tumor-selective drug accumulation, and decreased toxicity to normal tissues. The cell-surface transmembrane glycoprotein CD44 binds to the extracellular domain of hyaluronic acid (HA), and is overexpressed in breast, ovarian, lung, and stomach cancer. In this study, an HA-based nano-carrier incorporating doxorubicin (DOX) and cisplatin (CDDP) was synthesized as a CD44-targeting anti-cancer drug delivery system, and its tumor inhibition effects against CD44+ breast cancer cells were evaluated in vitro and in vivo. These dual drug-loaded HA micelles (HA-DOX-CDDP) exhibited significantly enhanced drug release under acidic conditions, and showed higher cellular uptake and stronger cellular growth inhibition than free drugs against 4T1 (CD44+) breast cancer cells. In contrast, no significant differences in growth inhibition and cellular uptake were observed between HA-DOX-CDDP and free drugs in NIH-3T3 (CD44-) control cells. Furthermore, HA-DOX-CDDP micelles exhibited stronger inhibitory effects and lower systemic toxicity than free drugs in a 4T1 mammary cancer-bearing mouse model, as determined using immunofluorescence and histological analyses. Therefore, HA-DOX-CDDP micelles represent a promising drug delivery system that exhibits acid-sensitive drug release, CD44-targeted delivery, and excellent biocompatibility and biodegradation. These properties resulted in excellent tumor accumulation and reduced adverse effects, indicating that HA-DOX-CDDP micelles have promising potential applications in chemotherapy for breast cancer.

Characterizing CD44 regulatory microRNAs as putative therapeutic agents in human melanoma.

The multistructural and multifunctional transmembrane glycoprotein CD44 is overexpressed in many tumors of distinct origin including malignant melanoma and contributes to a poor prognosis by affecting cell proliferation, cell migration, and also the sensitivity for apoptosis induction. Previous studies reported so far 15 CD44 regulatory microRNAs (miRs) in different cell systems.Using a novel method for miR affinity purification miR-143-3p was identified as most potent binder to the 3’ untranslated region (UTR) of CD44. Overexpression of miR-143-3p in melanoma cells inhibits CD44 translation, which is accompanied by a reduced proliferation, migration and enhanced daunorubicin induced apoptosis of melanoma cells in vitro.Analyses of discordant CD44 and miR-143-3p expression levels in human melanocytic nevi and dermal melanoma samples demonstrated medium to high CD44 levels with no association to tumor grading or staging. The CD44 expression correlated to PD-L1, but not to MART-1 expression in malignant melanoma. Interestingly, the CD44 expression was inversely correlated to the infiltration of pro-inflammatory immune effector cells.In conclusion, the tumor suppressive miR-143-3p was identified as the most potent CD44 inhibitory miR, which affects growth characteristics of melanoma cells suggesting the implementation of miR-143-3p as as a potential anti-CD44 therapy of malignant melanoma.

Immunome Single Cell Profiling Reveals T Cell Exhaustion with Upregulation of Checkpoint Inhibitors LAG3 and Tigit on Marrow Infiltrating T Lymphocytes in Daratumumab and IMiDs Resistant Patients

Background: The addition of immunomodulatory (IMiDs) drugs to monoclonal antibodies targeting the transmembrane glycoprotein CD38 has demonstrated very encouraging and durable responses in myeloma patients. It is believed that the synergistic effects observed with anti-CD38 antibodies and IMiDs are derived from their co-modulation of the host adaptive and innate immunity and therefore it is plausible to speculate that acquired resistance to daratumumab and IMiDs may be largely immune-mediated. The aim of the present study was to 1) interrogate at the single cell level the bone marrow immune repertoire of daratumumab sensitive and resistant patients, 2) identify cellular mediators of resistance to anti-CD38 antibodies and 3) define potential means to reinstate sensitivity to daratumumab and IMiDs.Methods and Results: Serial BM aspirates (n=44) were collected from patients treated with single agent daratumumab (MMY3012 trial) or daratumumab + pomalidomide (MM014 trial) prior to initiation of therapy, C3D1 and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD138pos and CD138neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Paired-end sequencing was performed on Illumina NEXTseq and NOVAseq platforms. Cell Ranger Single and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting and data analysis. Sequencing data were analyzed by principal component analysis (PCA), clustering with multi-sample batch correction and then visualized by t-distributed stochastic neighbor embedding (t-SNE) projection. Comparison of the single cell transcriptomes of CD138neg cells from responding patients pre- and post- treatment revealed that Daratumumab and Pomalidomide dramatically modify the immune cells composition (immunome) of the bone marrow niches leading to: 1) expansion of effector T cells (KLRG1high, GZMAhigh, CCL5high), 2) significant depletion of CD38high / FCGR3Ahigh NK cells with retained population of cytotoxic NK cells (CD27high, KLRB1high, NCR3high, GZMApos, PRF1pos), 3) depletion of FCGR3Ahigh / CD14low monocytes, 4) expansion of M1 inflammatory macrophages and depletion of plasmacytoid dendritic cells. Similar changes were seen in patients treated with single agent daratumumab (without IMiDs) however with a lesser expansion of effector T cells and in particular reduced marrow infiltrating inflammatory macrophages. In contrast, the immunome of daratumumab and pomalidomide resistant patients was characterized by a reduced central memory T cells (TCM), and a largely exhausted effector T cells populations that are CD28neg and expressing checkpoint inhibitors (LAG3and TIGIT significantly more than PDCD1) as well as high expression of TIM3 (HAVCR2) on marrow macrophages. Upregulation of LAG3 and TIGIT expression on T cells was also confirmed at the antigenic level by flow cytometry. Consistent with the non-bystander and suppressive effect of LAG3 and TIGIT on the function of effector T cells, activation (CD107a expression) and proliferation of LAGpos and/or TIGITpos sorted bone marrow T cells from resistant patients were significantly reduced in response to autologous myeloma cells stimulation or CD3/CD28 crosslinking. Lastly, a higher proportion and number of clonal T cell (through single cell TCR sequencing) was also observed in responding (≥ PR) vs non-responding (< PR) patients. Interrogation of the myeloma cells transcriptome, showed little to no loss of CD38 transcript at the time of acquired resistance with rather upregulation of complement inhibitory molecule CD59 and NFκB signature genes.Conclusion: A systematic unsupervised interrogation of the bone marrow immunome of daratumumab and IMiDs treated MM patients demonstrated a significant activation of adaptive and innate immunity in responding patients and revealed an expansion of exhausted T cells with upregulation of the checkpoint inhibitors LAG3 and TIGIT in resistant patients. Our findings warrant the exploration of LAG3- and/or TIGIT-blocking strategies as potential means to reinstate sensitivity to daratumumab and IMiDs in myeloma patients. DisclosuresNeri:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. McCulloch:Celgene: Honoraria; Takeda: Other: Travel expenses. Thakurta:Celgene Corporation: Employment, Equity Ownership. Bahlis:Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

Tunable conjugation densities of camptothecin on hyaluronic acid for tumor targeting and reduction-triggered release.

Micelles self-assembled from drug-conjugated polymers indicate advantages in alleviating the premature release before reaching the intended site. Hyaluronic acid (HA) is known to specifically bind with a transmembrane glycoprotein CD44, overexpressed in many types of cancerous cells, and can also be served as micelle carriers. However, an excess amount of drug conjugation to HA backbone may be detrimental to the receptor-mediated cellular uptake. Up to now, the effect of conjugation densities of drugs has never been determined on the physical properties and biological performance of resulting micelles. In the current study, camptothecin (CPT) was conjugated on HA through 3,3'-dithiodipropionic acid to self-assemble into reduction-sensitive micelles. The substitution degrees of CPT on HA backbone were tuned from around 4-20%, to clarify the effects on the cellular uptake efficiency and cytotoxicities of micelles, as well as the tumor accumulation and antitumor efficacy. The CPT substitution degree of around 15% on HA resulted in micelles with a higher cytotoxicity to 4T1 cells and achieved a better balance between the cellular uptake and reduction-triggered drug release, compared with other micelles. In contrast to a fast kidney clearance and an even distribution in major organs after intravenous injection of free CPT, the optimized micelles were accumulated in tumors, livers and lungs. The micelle content indicated a significant decrease in livers after 24h, while that in tumors displayed a significant increase to 4.9% of the injection dose. The tumor accumulation of micelles led to strong tumor suppression with minimal systemic toxicity. The in situ tumor inhibition and the accumulation of micelles in liver and lungs inhibited tumor metastasis to these tissues. It demonstrates a feasible strategy to develop drug-HA conjugate micelles with a concise and tunable structure for tumor targeting and reduction-triggered release. Hyaluronic acid (HA) can be served as micelle carriers and targeting ligands to tumor cells. However, the effects of drug conjugation densities on the physical profile and biological performance of resulting micelles have never been investigated. In the current study, camptothecin is conjugated on HA with reduction-sensitive linkers, and the substitution degrees of camptothecin on HA backbone vary from around 4-20%. The micelles with a substitution degree of around 15% achieve a better balance between the cellular uptake and reduction-triggered drug release and a higher cytotoxicity than others. It demonstrates a feasible strategy to develop drug-HA conjugate micelles with a concise and tunable structure for tumor targeting and reduction-triggered release.

Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent.

BackgroundAlthough epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation.MethodsWild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures.ResultsIn WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice.ConclusionWe demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.

The regulation of CD44 expression by new tumor suppressor gene Arid2 and the influence of Arid2 on the invasion and metastasis in hepatocellular carcinoma cells

To investigate the effect of a new tumor-suppressor gene Arid2 on the expression level of CD44 and the mechanism that Arid2 regulates the invasion and metastasis in human hepatocellular carcinoma cells HepG2 and Huh7. Recombinant pGL3-CD44 reporter plasmids were transfected into hepatoma cell lines HepG2 and Huh7 cells infected with adenovirus Arid2(Ad-Arid2). Dual luciferase assays were used to determine the relative luciferase activities of reporter plasmids. Western blot technique was used to detect the influence of Arid2 on the expression of transmembrane glycoprotein CD44. Cell migration assays of tumor cells were employed to observe the impact of overexpression of Arid2 on the invasion and metastasis abilities of tumor cells. The sizes of transplanted tumors were recorded to observe the growth of subcutaneous transplanted tumors in nude mice. Statistical significance was analyzed by one-way ANOVA for multiple comparisons, and independent-samples t-test was utilized to compare two groups. Luciferase assay showed cells were transfected with different length of CD44 reporter plasmids, and their relative luciferase activities were improved to different degrees, compared with pGL3-Basic control. Meanwhile, the mean luciferase activities of pGL3-CD44 -791~+224bp reporter plasmids were significantly repressed by the overexpression of Arid2 which inhibition rates were up to 73.83%±0.92%(P< 0.05, HepG2) or 48.99%±1.37% (P <0.05, Huh7), compared with Ad-GFP control. Western blot results showed that CD44 protein expression was obviously decreased by overexpression of Arid2. Cell migration assays confirmed that the invasion and metastasis abilities were inhibited by increasing Arid2 expression in Human HepG2 or Huh7 cells, which inhibition rates were 66.95%±0.59%(P< 0.05)in HepG2 cells and 73.86%±0.49%(P< 0.05) in Huh7 cells respectively. The animal experiment results indicated that Arid2 could obviously delay or restrict the subcutaneous transplanted tumors growth in nude mice, which was declined by 98.57%±0.34%(P< 0.05). CD44 promoter activities and protein expressions were significantly down-regulated by Arid2 in vitro. The growth and metastasis of tumors were obviously restrained in the hepatocellular carcinoma cells and nude models. In brief, these researches indicate CD44 may play important roles in the process where the invasion and metastasis of hepatocellular carcinoma cells are under the control of Arid2.The studies introduce and evaluate the relationships between Arid2 and CD44, and further provide a new research direction with the occurrence and development of hepatocellular carcinoma.

SYK Is Involved in the CD38 Signal Transduction Pathway in Chronic Lymphocytic Leukemia

B-cell chronic lymphocytic leukemia (CLL) is one of the most prevalent B cell malignancies in adults and characterized by expansion of monoclonal mature B cells. Survival and proliferation of CLL cells depends on microenvironmental contact in lymphoid organs. The transmembrane glycoprotein CD38 acts as an important mediator of survival, proliferation and migration signals for CLL cells, and its expression is associated with poor prognosis. Spleen tyrosine kinase (SYK) is a central element of the B-cell receptor signal transduction pathway and has additionally been shown to be involved in cytokine and integrine signaling. In this study we demonstrate direct involvement of SYK in the CD38 signaling pathway in primary CLL samples.CD38-stimulation of primary CLL cells by its ligand CD31 induced a consistent phosphorylation of the tyrosine residue Y352, the first activation site that releases SYK from its autoinhibitory conformation (p<0.001). SYK downstream targets AKT (p<0.05) and ERK (p<0.05) were subsequently induced and prolonged CD38-stimulation increased MCL-1-expression (p<0.05). Concomitant inhibition of SYK with the SYK inhibitor R406 resulted in inhibition of AKT- and ERK-activation (p<0.05 and p<0.01) and prevented upregulation of MCL-1 (p<0.01). Moreover, we observed SYK-dependent enhancement of BCR-signaling after CD38 ligation. Short-term exposure of CLL cells to CD31 led to an increase of ERK-phosphorylation after BCR-engagement by 41.9% (p<0.05). This effect was completely abolished by concomitant R406-treatment (p<0.05). Additionally, we observed a SYK-dependent increase of Ca2+-flux in response to BCR-stimulation after previous CD38 activation. Moreover, preliminary experiments show that prolonged CD38-stimulation led to a SYK-dependent increase of baseline Ca2+-flux in CLL cells, indicating a potential involvement of CD38 in autonomous BCR-signaling. CD38 acts as an enhancer of migratory stimuli in CLL cells. We therefore analysed, whether SYK is also involved in this interaction process. CXCL12-dependent migration was increased by CD38 stimulation with the agonistic CD38 antibody IB4 by 28.3% (p=0.12). Treatment of CLL cells with R406 completely inhibited IB4-mediated migration (p<0.01). The expression of CD38 is regulated by a variety of mechanisms, including CD40 ligation. SYK is involved in CD40 signaling. We therefore tested, whether SYK-inhibition affects CD38-expression. Stimulation of CLL cells with recombinant CD40L resulted in a significant increase of CD38-expression (p<0.05). This effect was reversed by concomitant SYK-inhibition (p<0.01). In addition, we observed marked down-regulation of CD38 surface-expression (p<0.05) and mRNA-expression (p<0.05) for CLL cells treated with SYK-inhibitors R406 or P505-15 compared to vehicle control. This effect is at least partly based on transcriptional inhibition of CD38-regulators NF-kB (p<0.05) and E2A (p<0.05). Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy (p<0.01).In conclusion, our data show that SYK acts as a central downstream effector of CD38 signaling regulating survival-, proliferation-, and migration pathways in CLL and also functions as a strong regulator of CD38 expression. The interaction of CD38 and SYK involves the BCR pathways, where CD38 enhances the response to BCR-stimulation and, moreover, may act as an enhancer of autonomous BCR-signaling in CLL. Additionally, the CD38-SYK interaction involves BCR-independent microenvironmental pathways as shown for CD40 and CXCL12. CD38 expression not only serves as a negative prognostic marker but has also been shown to possess biological significance in CLL. We therefore propose that disruption of the CD38-SYK axis may represent a promising therapeutic option in CLL. DisclosuresNo relevant conflicts of interest to declare.

The Prophage-encoded Hyaluronate Lyase Has Broad Substrate Specificity and Is Regulated by the N-terminal Domain

Streptococcus equi is the causative agent of the highly contagious disease "strangles" in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded β-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection.