scholarly journals The Calcitriol/Vitamin D Receptor System Regulates Key Immune Signaling Pathways in Chronic Lymphocytic Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 285
Author(s):  
Marina Gerousi ◽  
Fotis Psomopoulos ◽  
Konstantia Kotta ◽  
Maria Tsagiopoulou ◽  
Niki Stavroyianni ◽  
...  
Keyword(s):  
Vitamin D ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Ex Vivo ◽  
Active Form ◽  
Lymphocytic Leukemia ◽  
Biologically Active ◽  
Sequencing Analysis ◽  
Receptor System

It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcomes in various B cell malignancies, including chronic lymphocytic leukemia (CLL). In this study, we sought to obtain deeper biological insight into the role of vitamin D and its receptor (VDR) in the pathophysiology of CLL. To this end, we performed expression analysis of the vitamin D pathway molecules; complemented by RNA-Sequencing analysis in primary CLL cells that were treated in vitro with calcitriol, the biologically active form of vitamin D. In addition, we examined calcitriol effects ex vivo in CLL cells cultured in the presence of microenvironmental signals, namely anti-IgM/CD40L, or co-cultured with the supportive HS-5 cells; and, CLL cells from patients under ibrutinib treatment. Our study reports that the calcitriol/VDR system is functional in CLL regulating signaling pathways critical for cell survival and proliferation, including the TLR and PI3K/AKT pathways. Moreover, calcitriol action is likely independent of the microenvironmental signals in CLL, since it was not significantly affected when combined with anti-IgM/CD40L or in the context of the co-culture system. This finding was also supported by our finding of preserved calcitriol signaling capacity in CLL patients under ibrutinib treatment. Overall, our results indicate a relevant biological role for vitamin D in CLL pathophysiology and allude to the potential clinical utility of vitamin D supplementation in patients with CLL.

scholarly journals Functional Calcitriol/Vitamin D Receptor Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3019-3019
Author(s):  
Marina Gerousi ◽  
Fotis Psomopoulos ◽  
Kostantia Kotta ◽  
Niki Stavroyianni ◽  
Achilles Anagnostopoulos ◽  
...  
Keyword(s):  
Vitamin D ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Cell Line ◽  
Vitamin D Receptor ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Biologically Active ◽  
Differentially Expressed ◽  
Rna Seq

Calcitriol, the biologically active form of vitamin D, modulates a plethora of cellular processes following its receptor ligation, namely the vitamin D receptor (VDR), a nuclear transcription factor that regulates the transcription of diverse genes. It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcome in chronic lymphocytic leukemia (CLL). Recently, we reported that VDR is functional in CLL cells after calcitriol supplementation, as well as after stimulation through both the calcitriol/VDR signaling system and other prosurvival pathways triggered from the tumor microenvironment. In this study, we aimed at investigating key molecules and signaling pathways that are altered after calcitriol treatment and are known to play a relevant role in CLL pathophysiology. CD19+ primary CLL cells were negatively selected from peripheral blood samples of patients that were treatment naïve at the time of sample collection. CLL cells were cultured in vitro with calcitriol or co-cultured with the HS-5 mesenchymal cell line for 24 hours. VDR+, CYP24A1+, phospho-ERK+ and phospho-NF-κB p65+ cells were determined by Flow Cytometry (FC). Total RNA was extracted from calcitriol-treated and non-treated CLL cells, while mRNA selection was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation for RNA-Sequencing (RNA-Seq) analysis was conducted with the NEBNext Ultra II Directional RNA Library Prep Kit. The libraries were paired-end sequenced on the NextSeq 500 Illumina platform. Differential expression analysis was performed using DESeq2; genes with log2FC>|1| and P≤0.05 were considered as differentially expressed. RNA-Seq analysis (n=6) confirmed our previous findings that the CYP24A1 gene is significantly upregulated by calcitriol, being the top upregulated gene, whereas the VDR gene remains unaffected by this treatment. Overall, 85 genes were differentially expressed in unstimulated versus calcitriol-treated cells, of which 28 were overexpressed in the latter thus contrasting the remaining 57 which showed the opposite pattern. Pathway enrichment and gene ontology (GO) analysis of the differentially expressed genes revealed significant enrichment in PI3K-Akt pathway and Toll-like receptor cascades, as well as in vitamin D metabolism and inflammatory response pathways. Additionally, flow cytometric analysis showed that calcitriol-treated CLL cells displayed increased pERKlevels (FD=1.3, p<0.05) and, in contrast decreased pNF-κBlevels (FD=2.7, p<0.05), highlighting active VDR signaling in CLL. Aiming at placing our findings in a more physiological context, we co-cultured CLL cells with the HS-5 cell line. Based on our previous finding that co-cultured CLL cells showed induced CYP24A1 levels, we evaluated pNF-κB expression. pNF-κB levels were found to be increased in co-cultured CLL cells (FD=4.2, p<0.05), while the addition of calcitriol downregulated pNF-κB (FD=1.5, p<0.05). Moreover, ex vivo calcitriol exposure of CLL cells from patients under ibrutinib treatment (at baseline, +1 and +3-6 months, n=7) resulted in significant upregulation of pERK (FD=1.6, p<0.01; FD=1.4, p<0.01; FD=1.9, p<0.01; for each timepoint respectively) but, significant downregulation of pNF-κΒ (FD=3.4, p<0.01; FD=3, p<0.05; FD=2.3, p<0.05; for each timepoint respectively), indicating preserved calcitriol/VDR signaling capacity. In conclusion, we provide evidence that the calcitriol/VDR system is active in CLL, modulating NF-κB and MAPK signaling as well as the expression of the CYP24A1 target gene. This observation is further supported by RNA-Seq analysis that confirms CYP24A1 upregulation and highlights new signaling pathways that need to be validated. Interestingly, the calcitriol/VDR system appears relatively unaffected by either stimulation or inhibition (ibrutinib) of microenvironmental signals that promote CLL cell survival and/or proliferation, indicating context-independent signaling capacity. Disclosures Kotsianidis: Celgene: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

scholarly journals Vitamin D alleviates oxidative stress in adipose tissue and mesenteric vessels from obese patients with subclinical inflammation

2020 ◽  
Vol 98 (2) ◽  
pp. 85-92 ◽  
Author(s):  
Mihaela Ionica ◽  
Oana M. Aburel ◽  
Adrian Vaduva ◽  
Alexandra Petrus ◽  
Sonia Rațiu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Vitamin D ◽  
Adipose Tissue ◽  
Vascular Function ◽  
Ex Vivo ◽  
Active Form ◽  
Reactive Oxygen Species Generation ◽  
Obese Patients ◽  
Tissue Samples

Obesity is an age-independent, lifestyle-triggered, pandemic disease associated with both endothelial and visceral adipose tissue (VAT) dysfunction leading to cardiometabolic complications mediated via increased oxidative stress and persistent chronic inflammation. The purpose of the present study was to assess the oxidative stress in VAT and vascular samples and the effect of in vitro administration of vitamin D. VAT and mesenteric artery branches were harvested during abdominal surgery performed on patients referred for general surgery (n = 30) that were randomized into two subgroups: nonobese and obese. Serum levels of C-reactive protein (CRP) and vitamin D were measured. Tissue samples were treated or not with the active form of vitamin D: 1,25(OH)2D3 (100 nmol/L, 12 h). The main findings are that in obese patients, (i) a low vitamin D status was associated with increased inflammatory markers and reactive oxygen species generation in VAT and vascular samples and (ii) in vitro incubation with vitamin D alleviated oxidative stress in VAT and vascular preparations and also improved the vascular function. We report here that the serum level of vitamin D is inversely correlated with the magnitude of oxidative stress in the adipose tissue. Ex vivo treatment with active vitamin D mitigated obesity-related oxidative stress.

Preclinical Evaluation of the Proteasome Inhibitor MLN9708 (MLN2238) In Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4634-4634
Author(s):  
Aisha Masood ◽  
Kiersten M Miles ◽  
Nazmul H Khan ◽  
Drusilla Akhtar ◽  
Remi Adelaiye ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Proteasome Inhibitor ◽  
Active Form ◽  
Lymphocytic Leukemia ◽  
Biologically Active ◽  
Autologous Serum ◽  
Mitochondrial Outer Membrane ◽  
Apoptosis Pathway ◽  
Intrinsic Apoptosis Pathway

Abstract Abstract 4634 The proteasome is an important therapeutic target in multiple hematological malignancies. The proteasome inhibitor bortezomib has demonstrated significant clinical activity in multiple myeloma and mantle cell lymphoma. However, clinically bortezomib has failed to demonstrate efficacy in chronic lymphocytic leukemia (CLL); the exact reason for this remains unknown. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a proteasome inhibitor which has a shorter proteasome dissociation half life than bortezomib and is currently in Phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all of the studies reported here, in which we evaluated the antileukemic effects of MLN2238 in primary CLL cells from 16 patients. MLN 2238 induced a time and dose dependent decrease in CLL cell viability in 12 (75%) patient cells in vitro. Cell death was shown to be due to the induction of apoptosis confirmed by Annexin V staining of CLL cells and cleavage of PARP-1, an indicator of apoptosis. Biochemical analysis showed that caspase 3 and 9 were activated in these cells, indicating that MLN2238 induces cell death through the intrinsic apoptosis pathway. We noted increased mitochondrial outer membrane permeability (MOMP) in the presence of MLN2238, which suggests the engagement of the mitochondria in MLN2238 induced apoptosis in CLL cells. Evaluation of non-responding CLL patient samples demonstrated that increased expression of PSMB5 is associated with lack of sensitivity to MLN2238. Furthermore, incremental addition of autologous serum in responding CLL samples resulted in upregulation of PSMB5 levels, resulting in protection against MLN2238 induced cell death. Our preclinical observations demonstrate for the first time that MLN2238 can induce death in primary CLL cells, and support further investigation in CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 536-543
Author(s):  
GB Faguet ◽  
JF Agee
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Fab Fragment ◽  
In Vitro Characterization ◽  
The Common ◽  
Dose Dependent

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.

scholarly journals Vitamin D Enhances Radiosensitivity of Colorectal Cancer by Reversing Epithelial-Mesenchymal Transition

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinyue Yu ◽  
Qian Wang ◽  
Baocai Liu ◽  
Ning Zhang ◽  
Guanghui Cheng
Keyword(s):  
Colorectal Cancer ◽  
Vitamin D ◽  
Epithelial Mesenchymal Transition ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Biologically Active ◽  
Plasminogen Activator Inhibitor 1 ◽  
Mesenchymal Transition

Colorectal cancer (CRC) is often resistant to conventional therapies. Previous studies have reported the anticancer effects of vitamin D in several cancers, its role in radiotherapy (RT) remains unknown. We found that 1α, 25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, had antitumor effect on CRC and sensitized CRC cells to ionizing radiation (IR). VD3 demonstrated synergistic effect in combination with IR, which were detected by colony formation and cell proliferation assay. Radiosensitivity restoration induced by VD3 was associated with a series of phenotypes, including apoptosis, autophagy, and epithelial-mesenchymal transition (EMT). Using proteomics, “regulation of cell migration” and “cadherin” were found to be obviously enriched GO terms. Moreover, cystatin D and plasminogen activator inhibitor-1 (PAI-1), the differentially expressed proteins, were associated with EMT. Next, we confirmed the contributions of these two genes in enhancing IR sensitivity of CRC cells upon inhibition of EMT. As determined by proteomics, the mechanism underlying such sensitivity involved partially block of JAK/STAT3 signaling pathway. Furthermore, VD3 also elicited sensitization to RT in xenograft CRC models without additional toxicity. Our study revealed that VD3 was able to act in synergy with IR both in vitro and in vivo and could also confer radiosensitivity by regulating EMT, thereby providing a novel insight for elevating the efficacy of therapeutic regimens.

scholarly journals Vegetal diamine oxidase alleviates histamine-induced contraction of colonic muscles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Armelle Tchoumi Neree ◽  
Rodolphe Soret ◽  
Lucia Marcocci ◽  
Paola Pietrangeli ◽  
Nicolas Pilon ◽  
...  
Keyword(s):  
Diamine Oxidase ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Active Form ◽  
Distal Colon ◽  
Biologically Active ◽  
In Vitro Assays ◽  
Histaminase Activity ◽  
Irritable Bowel

AbstractExcess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5′-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.

The Mevalonate Pathway and Downstream Signal Transducers As Therapeutic Targets to Overcome Multidrug Resistance in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3881-3881
Author(s):  
Marta Coscia ◽  
Micol Rigoni ◽  
Chiara Riganti ◽  
Candida Vitale ◽  
Ivana Campia ◽  
...  
Keyword(s):  
Cell Death ◽  
Multidrug Resistance ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Stromal Cells ◽  
Active Form ◽  
Rho Kinase ◽  
Lymphocytic Leukemia ◽  
Pathway Activity ◽  
Downstream Signaling

Abstract Abstract 3881 Background: The mutational status of tumor immunoglobulin heavy chain variable region (IGHV) is a reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. The tumor microenvironment actively supports the survival of CLL cells and confers a multidrug resistance (MDR) phenotype to CLL cells. MDR is due to the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. Pgp is under the positive control of the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α) which is activated by isoprenylated Ras/Rho-dependent downstream signaling pathways. Ras and Rho isoprenylation are regulated by the mevalonate (Mev) pathway activity suggesting that this pathway can be exploited as a metabolic checkpoint to regulate chemresistance. Aim: The aim of this study was twofold: 1) to investigate the correlation between chemoresistance and the activity of the Mev pathway and Ras/Rho-A downstream signaling pathways in purified M and UM CLL cells under basal conditions and after incubation with stromal cells; 2) to evaluate the chemosensitizing effects of agents specifically targeting the Mev pathway and downstream signaling pathways under the same culture conditions. Methods: M and UM CLL cells were cultured in the presence and in the absence of murine stromal cells (M210B4) and exposed to Zoledronic acid (ZA) (1 μmol/L), Simvastatine (Sim) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L), HIF-1α inhibitor YC-1 (10 μmol/L) and Doxorubicine (Doxo) (1 μmol/L). The Mev pathway activity was measured by cells radiolabelling with [14C]-mevalonic acid and thin layer chromatography. Ras, ERK1/2 and Akt activity were detected by Western blot. Rho, Rho Kinase and HIF-1α activity were assessed by ELISA. Mdr1 expression was measured by Real Time-PCR. PgP activity was evaluated by measuring Doxo intracellular accumulation. Doxo cytotoxicity was assessed by annexin V and propidium iodide staining. Results: The Mev pathway is significantly more active in UM than in M CLL cells. This hypermetabolic activity translates into a higher activation of Ras/Akt and Rho/Rho kinase signaling pathways and higher expression of the phosphorylated active form of HIF-1α. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to a more effective Doxo extrusion and therefore better survival upon Doxo exposure. M210B4 stromal cells further protect UM CLL cells from Doxo induced cell death by upregulating Mev pathway activity, HIF-1α/mdr1/PgP axis activation, and Doxo extrusion. Targeting the Mev pathway of UM cells with ZA and Mev reduces the basal activity of HIF-1α/mdr1/PgP axis and significantly increases Doxo retention and cytotoxicity. Similar effects are obtained with PD85 and YC1–10 which are specific inhibitors of the downstream molecules ERK-1/2 and HIF-1α, respectively. All these agents are able to overcome the protective effect exerted by stromal cells by significantly increasing PgP activity and Doxo-induced cell death. Conclusions: Our data demonstrate that the Ras- and Rho-dependent HIF-1α/mdr1/PgP axis is more active and associated with higher levels of MDR in UM compared with M CLL cells. Targeting the Mev pathway and/or downstream signalling pathways is a promising strategy to circumvent basal and stroma-mediated chemoresistance especially in UM CLL cells. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding.

scholarly journals Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression

2021 ◽  
Vol 11 (7) ◽  
Author(s):  
Miguel Quijada-Álamo ◽  
María Hernández-Sánchez ◽  
Ana-Eugenia Rodríguez-Vicente ◽  
Claudia Pérez-Carretero ◽  
Alberto Rodríguez-Sánchez ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nuclear Translocation ◽  
Ex Vivo ◽  
Biological Significance ◽  
Xenograft Model ◽  
Underlying Disease ◽  
Lymphocytic Leukemia ◽  
Enhanced Sensitivity

AbstractBIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.

TLR7/8 Agonists Overcome the Suppression of FcγR Activity in Monocytes From Chronic Lymphocytic Leukemia Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4595-4595
Author(s):  
Jonathan P. Butchar ◽  
Prexy Shah ◽  
Hemal Patel ◽  
Kavin Fatehchand ◽  
Sarwish Rafiq ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Cytokine Production ◽  
Treated Patient ◽  
Antibody Therapy ◽  
Lymphocytic Leukemia ◽  
Therapeutic Antibody ◽  
Receptor Modulation ◽  
Selective Agonist

Abstract Abstract 4595 It has been documented that monocytes from Chronic Lymphocytic Leukemia (CLL) patients are immunosuppressed (Flieger et al., 1990), and this may play a significant role in attenuating responses to antibody therapy. Fc-gamma receptors (FcγR) are critical for binding therapeutic antibody and effecting tumor cell destruction. Both monocytes and Natural Killer (NK) cells have been shown to be critical for this process. FcγR cluster upon binding opsonized targets and this results in activation of downstream mediators such as Ras and PI3K. These activation events are tempered, however, both at the receptor level and within the signaling pathways. The inhibitory FcγR, FcγRIIb, clusters along with the activating FcγR and dampens signaling. This receptor recruits SHIP, an inositol phosphatase that antagonizes PI3K. Other phosphatases such as SHIP2 and SHP-1 also serve to negatively regulate FcγR activity. Toll-like receptors (TLR) are innate immune receptors that recognize characteristic components of pathogens and other potentially harmful agents. Recently we discovered that treatment of human monocytes with the TLR7/8 agonist R-848 led to enhanced antitumor activity of therapeutic antibody in vitro, and in a murine solid-tumor model of antibody therapy. Hence, we began to examine the extent of suppression within the FcγR pathway of CLL patient monocytes, with the goal of testing whether TLR7/8 activation could at least in part reverse this suppression. We first tested FcγR-mediated cytokine responses and found they were significantly attenuated in freshly isolated monocytes from CLL patients. However, real-time PCR showed equivalent levels of FcγR in monocytes from patients and healthy donors, suggesting that the suppression was occurring in one or more portions of the downstream signaling pathways. We then examined phosphatase expression and found increased levels of SHIP, SHIP2 and SHP-1 in CLL patient monocytes. Next, we tested the functional effects of TLR7/8 agonist treatment on monocytes from CLL patients. Overnight treatment with 1 μM R-848 led to dramatic and significant increases in FcγR-mediated cytokine production. To determine whether this would still occur in the presence of the immunosuppressive B-CLL cells, we treated patient PBMC with R-848 and rituximab. ELISA results showed significant enhancement of antibody-mediated cytokine production with R-848 treatment. To determine whether TLR7/8 activation could increase the antibody-mediated clearance of B-CLL cells we treated patient PBMC overnight with or without rituximab and with either R-848 or vehicle. Results showed an approximately twofold increase in B-CLL cell clearance with R-848. These findings suggest that TLR7/8 activation can serve to overcome the monocyte immunosuppression seen with CLL. We then examined changes in FcγR expression and found that R-848 increased the expression of activating FcγR while decreasing expression of the inhibitory FcγRIIb in patient monocytes. In conjunction with this receptor modulation, we found significant decreases in transcripts of SHIP, SHIP2 and SHP-1. These results show that TLR7/8 activation enhances FcγR expression and drives concomitant decreases in inhibitory phosphatase expression in CLL patient monocytes. Because we used an agonist that activated both TLR7 and TLR8, it was unclear which TLR was primarily responsible for the changes observed. Hence, we treated monocytes with 3M–055 (TLR7-selective), R-848 (TLR7/8 dual-agonist) or a TLR8-selective agonist and measured FcγR expression. Results showed that activation of both TLR7 and TLR8 was sufficient to drive upregulation of the activating FcγR. However, TLR7 activation alone had no effect on the inhibitory receptor FcγRIIb. Conversely, the TLR8-selective agonist led to an almost complete abolishment of FcγRIIb expression. Dual TLR7/8 activation decreased FcγRIIb to levels between TLR7- and TLR8-selective agonist treatment. We also found that all 3 agonists reduced phosphatase expression, with the TLR8-selective agonist leading to the greatest decrease. In summary, these results show that FcγR function but not expression is compromised in CLL patient monocytes. However, treatment with TLR7/8 agonists, in particular TLR8-selective agonists, can reverse this suppression and promote stronger FcγR responses. Hence, these compounds may be particularly useful as adjuvants for antibody therapy. Disclosures: Vasilakos: 3M: Employment.

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