Wnt inhibitors reduce the unfolded protein response and enhance bortezomib-induced cell death in multiple myeloma

The Wnt pathway is one of the main regulators of cell growth and is essential for embryonic development and tissue homeostasis.1 Dysregulation of the Wnt pathway is involved in the pathogenesis of many types of carcinomas and is associated with certain hematological cancers, including multiple myeloma (MM).2-4 Several studies, primarily using cell lines and mouse models, reported that MM plasma cells (PCs) carry a functional Wnt/β-catenin signaling pathway that promotes cell survival.3-7 Despite new and successful treatment options, MM remains incurable because of clinical, genetic, and transcriptomic heterogeneity, which ultimately results in therapy resistance and relapse. Thus, an unmet need exists to find new therapeutic targets and improve responses to approved drugs. In the canonical Wnt pathway, Wnt ligands bind and activate Frizzled receptors and LRP5/6 coreceptors to recruit components of the β-catenin destruction complex.1,3 Subsequent stabilization of nonphosphorylated β-catenin results in the transcription of Wnt target genes. Here, we targeted 2 pathway components to inhibit Wnt signaling in MM cells. Inhibition of tankyrase promotes degradation of β-catenin owing to stabilization of Axin, which is the concentration-limiting component of the β-catenin destruction complex.8 Inhibition of porcupine, an enzyme required for the secretion of all human Wnt ligands, was shown to reduce Wnt signaling in vitro and in a mouse model.9 We demonstrate that targeting Wnt signaling promotes cell death of patient-derived MM cells and downregulates genes involved in the unfolded protein response (UPR). Subsequently, we show that Wnt inhibitors enhance bortezomib (BTZ)-induced cell death in primary MM cells. First, we tested the functionality of tankyrase inhibitor (TNKSi) XAV939 and porcupine inhibitor (PORCi) C59 in MM cell lines. Single-drug exposure in MM1.S blocked β-catenin upregulation by Wnt3a-conditioned medium in case of TNKSi and downregulated β-catenin expression in case of PORCi (Figure 1A). Treatment with TNKSi caused only moderate apoptosis in MM1.S and L363, ranging from 0.2% to 3.9% specific apoptosis at 10 to 40 μM TNKSi and 3.9% to 29.1% specific apoptosis at 5 to 10 μM PORCi (supplemental Figure 1A). The inhibitor combination induced more than additive cell death in MM1.S, with specific apoptosis increasing to 57.1% for 20 μM TNKSi and 10 μM PORCi (supplemental Figure 1A,B). A significant impact on the number of viable cells was observed after 7 days of treatment (supplemental Figure 1C). We also demonstrated a significant reduction in transcriptional Wnt reporter activity after treatment with both inhibitors (Figure 1B). In addition to MM1.S and L363, the combination of Wnt inhibitors was effective in MM cell lines INA-6 and KMS12- PE (supplemental Figure 1D). In summary, these data show that the Wnt pathway can be inhibited by a combination of TNKSi and PORCi and affects cell survival in MM cell lines. The Wnt pathway is one of the main regulators of cell growth and is essential for embryonic development and tissue homeostasis.1 Dysregulation of the Wnt pathway is involved in the pathogenesis of many types of carcinomas and is associated with certain hematological cancers, including multiple myeloma (MM).2-4 Several studies, primarily using cell lines and mouse models, reported that MM plasma cells (PCs) carry a functional Wnt/β-catenin signaling pathway that promotes cell survival.3-7 Despite new and successful treatment options, MM remains incurable because of clinical, genetic, and transcriptomic heterogeneity, which ultimately results in therapy resistance and relapse. Thus, an unmet need exists to find new therapeutic targets and improve responses to approved drugs. In the canonical Wnt pathway, Wnt ligands bind and activate Frizzled receptors and LRP5/6 coreceptors to recruit components of the β-catenin destruction complex.1,3 Subsequent stabilization of nonphosphorylated β-catenin results in the transcription of Wnt target genes. Here, we targeted 2 pathway components to inhibit Wnt signaling in MM cells. Inhibition of tankyrase promotes degradation of β-catenin owing to stabilization of Axin, which is the concentration-limiting component of the β-catenin destruction complex.8 Inhibition of porcupine, an enzyme required for the secretion of all human Wnt ligands, was shown to reduce Wnt signaling in vitro and in a mouse model.9 We demonstrate that targeting Wnt signaling promotes cell death of patient-derived MM cells and downregulates genes involved in the unfolded protein response (UPR). Subsequently, we show that Wnt inhibitors enhance bortezomib (BTZ)-induced cell death in primary MM cells. First, we tested the functionality of tankyrase inhibitor (TNKSi) XAV939 and porcupine inhibitor (PORCi) C59 in MM cell lines. Single-drug exposure in MM1.S blocked β-catenin upregulation by Wnt3a-conditioned medium in case of TNKSi and downregulated β-catenin expression in case of PORCi (Figure 1A). Treatment with TNKSi caused only moderate apoptosis in MM1.S and L363, ranging from 0.2% to 3.9% specific apoptosis at 10 to 40 μM TNKSi and 3.9% to 29.1% specific apoptosis at 5 to 10 μM PORCi (supplemental Figure 1A). The inhibitor combination induced more than additive cell death in MM1.S, with specific apoptosis increasing to 57.1% for 20 μM TNKSi and 10 μM PORCi (supplemental Figure 1A,B). A significant impact on the number of viable cells was observed after 7 days of treatment (supplemental Figure 1C). We also demonstrated a significant reduction in transcriptional Wnt reporter activity after treatment with both inhibitors (Figure 1B). In addition to MM1.S and L363, the combination of Wnt inhibitors was effective in MM cell lines INA-6 and KMS12- PE (supplemental Figure 1D). In summary, these data show that the Wnt pathway can be inhibited by a combination of TNKSi and PORCi and affects cell survival in MM cell lines

Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases

AV-block and conduction slowing prevail over TdP arrhythmias in the methoxamine-sensitized pro-arrhythmic rabbit model

INTRODUCTION: The methoxamine-sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization-dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in-house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances. METHODS AND RESULTS: Rabbits were randomly assigned to 3 groups: (1) 35 mg/kg ketamine + 5 mg/kg xylazine; (2) 0.5 mL/kg hypnorm + 3 mg/kg midazolam; (3) 35 mg/kg ketamine + 20 mg/kg propofol. Anesthesia was maintained by 1.5% isoflurane. Concomitant infusion of methoxamine (17 μg/kg/min for 40 minutes) and dofetilide (10 μg/kg/min for 30 minutes) was used to induce arrhythmias. Sole methoxamine infusion exclusively decreased HR in groups 1 and 3. Dofetilide lengthened repolarization, followed in time by PQ/QRS prolongation, second-degree AV block, and subsequently TdP arrhythmias. TdP was seen in 80%, 0%, and 33% of the rabbits in groups 1, 2, and 3, respectively. Decreasing the dose of dofetilide to 5 μg/kg/min in ketamine/xylazine anesthetized rabbits resulted in a drop in TdP incidence (25%) while conduction disturbances persisted. Flunarizine (n = 6) suppressed all TdP arrhythmias while conduction disturbances remained present. CONCLUSION: TdP incidence in the methoxamine-sensitized rabbit could be dramatically influenced by anesthesia, drug dose, and flunarizine, while conduction slowing remained present. Thus, conduction slowing seems to be the integral outcome in this model

AV-block and conduction slowing prevail over TdP arrhythmias in the methoxamine-sensitized pro-arrhythmic rabbit model

INTRODUCTION: The methoxamine-sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization-dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in-house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances. METHODS AND RESULTS: Rabbits were randomly assigned to 3 groups: (1) 35 mg/kg ketamine + 5 mg/kg xylazine; (2) 0.5 mL/kg hypnorm + 3 mg/kg midazolam; (3) 35 mg/kg ketamine + 20 mg/kg propofol. Anesthesia was maintained by 1.5% isoflurane. Concomitant infusion of methoxamine (17 μg/kg/min for 40 minutes) and dofetilide (10 μg/kg/min for 30 minutes) was used to induce arrhythmias. Sole methoxamine infusion exclusively decreased HR in groups 1 and 3. Dofetilide lengthened repolarization, followed in time by PQ/QRS prolongation, second-degree AV block, and subsequently TdP arrhythmias. TdP was seen in 80%, 0%, and 33% of the rabbits in groups 1, 2, and 3, respectively. Decreasing the dose of dofetilide to 5 μg/kg/min in ketamine/xylazine anesthetized rabbits resulted in a drop in TdP incidence (25%) while conduction disturbances persisted. Flunarizine (n = 6) suppressed all TdP arrhythmias while conduction disturbances remained present. CONCLUSION: TdP incidence in the methoxamine-sensitized rabbit could be dramatically influenced by anesthesia, drug dose, and flunarizine, while conduction slowing remained present. Thus, conduction slowing seems to be the integral outcome in this model

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic “knockout” models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment

Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K(IR)2.x carried I(K1) current and that inhibiting protein degradation in the lysosome increased intracellular K(IR)2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K(IR)2.1 to further restore normal levels of functional K(IR)2.1 channels. K(IR)2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K(IR)2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K(IR)2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I(K1) (at -60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute I(K1) block. Baf and CQ did not significantly enhance I(K1) densities. Pentamidine (10 μM, 48 h) reduced K(IR)2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K(IR)2.1 degradation. Pentamidine-induced downregulation of K(IR)2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued

Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis

Objectives Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology.Methods In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre: Runx3(f/f) mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model.Results Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis.Conclusions We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease

CXCL4 drives fibrosis by promoting several key cellular and molecular processes

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α

Objective. SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production.Methods. Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1 alpha and HIF-2 alpha gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays.Results. CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P &lt; 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2 alpha (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs.Conclusion. TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2 alpha pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4