Complex association of body mass index and outcomes in patients with relapsed and refractory multiple myeloma treated with CAR-T cell immunotherapy.

BACKGROUND AIMS: Chimeric antigen receptor-T (CAR-T) cells have exhibited remarkable efficacy in treating refractory or relapsed multiple myeloma (R/R MM). Although obesity has a favorable value in enhancing the response to immunotherapy, less is known about its predictive value regarding the efficacy and prognosis of CAR-T cell immunotherapy. METHODS: We conducted a retrospective study of 111 patients with R/R MM who underwent CAR-T cell treatment. Using the body mass index (BMI) classification, the patients were divided into a normal-weight group (73/111) and an overweight group (38/111). We investigated the effect of BMI on CAR-T cell therapy outcomes in patients with R/R MM. RESULTS: The objective remission rates after CAR-T cell infusion were 94.7% and 89.0% in the overweight and normal-weight groups, respectively. The duration of response and overall survival were not significant difference between BMI groups. Compared to normal-weight patients, overweight patients had an improved median progression-free survival. There was no significant difference in cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome between the subgroups. In terms of hematological toxicity, the erythrocyte, hemoglobin, platelet, leukocyte and neutrophil recovery was accelerated in the overweight group. Fewer patients in the overweight group displayed moderate percent CD4 and CD4/CD8 ratios compared to the normal-weight group. Furthermore, the percent CD4 ratios were positively correlated with the levels of cytokines [interleukin-2 (IL-2) (day 14), interferon gamma (IFN-γ) (day 7) and tumor necrosis factor alpha (TNF-α) (days 14 and 21)] after cells infusion. On the other hand, BMI was positively associated with the levels of IFN-γ (day 7) and TNF-α (days 14 and 21) after CAR-T cells infusion. CONCLUSIONS: Overall, this study highlights the potential beneficial effect of a higher BMI on CAR-T cell therapy outcomes.

Notch1 regulates hepatic thrombopoietin production.

Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, a role for Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency, and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control mice and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO mRNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-seq analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR) (asialoglycoprotein receptor 1, ASGR1) physically associates with Notch1 and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Dll4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed/refractory multiple myeloma: a phase I/II trial.

Despite the high therapeutic response achieved with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapy in relapsed and refractory multiple myeloma (R/R MM), primary resistance and relapse exist with single-target immunotherapy. Here, we design bispecific BC19 CAR T cells targeting BCMA/CD19 and evaluate antimyeloma activity in vitro and in vivo. Preclinical results indicate that BC19 CAR specifically recognize target antigens, and BC19 CAR T cells mediate selective killing of BCMA or CD19-positive cancer cells. BC19 CAR T cells also exhibit potent antigen-specific anti-tumor activity in xenograft mouse models. We conduct an open-label, single-arm, phase I/II study of BC19 CAR T cells in 50 patients with R/R MM (ChiCTR2000033567). The primary endpoint was safety. BC19 CAR T cells are well tolerated with grade 3 or higher cytokine release syndrome in 8% of patients and grade 1 neurotoxic events in 4% of patients, which meet the pre-specified primary endpoint. Secondary endpoints include overall response rate (92%), median progression-free survival (19.7 months), median overall survival (19.7 months) and median duration of response (not reached). Our study demonstrates that bispecific BC19 CAR T cells are feasible, safe and effective in treating patients with R/R MM.

Macrophage depletion damages hematopoiesis partially through inhibition of cell homing and expansion after hematopoietic cell transplantation.

Bone marrow macrophages (Mφ) are essential components of the bone marrow niche that regulate the function of hematopoietic stem cells. Poor graft function and inhibition of hematopoietic production can result from abnormal macrophage function; however, the underlying mechanism is unclear. Clodronate liposomes (Clo-Lip) have been used widely to deplete macrophages and study their functions. Our previous results showed that Clod-Lip-mediated clearance of macrophages plays a vital role in regulating hematopoietic reconstruction after allogeneic hematopoietic cell transplantation (HCT). In this study, using an isogenic hematopoietic stem cell transplantation model, we found that Clod-Lip-mediated clearance of macrophages suppressed hematopoietic reconstruction by inhibiting the homing process of hematopoietic cells. We also demonstrated that macrophage depletion inhibited the direct supportive effect of macrophages on hematopoietic stem and progenitor cells and erythroid differentiation but promoted the production of megakaryocytic progenitors ex vivo. We showed that macrophages increase CD49e expression on hematopoietic stem and progenitor cells (HSPCs). However, CD49e inhibitors did not support the proliferative effect of macrophages on hematopoietic cells. In contrast, macrophage E-selectin/ intercellular cell adhesion molecule-1 (ICAM-1) may be involved in directly regulating HSPCs. In conclusion, macrophage depletion with Clo-Lip partially disrupts bone marrow hematopoiesis after HCT by impeding donor cell homing and macrophage-HSPCs interactions.

Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution.

Bone marrow ablation is routinely performed before hematopoietic stem cell transplantation (HSCT). Hematopoietic stem and progenitor cells (HSPCs) require a stable bone marrow microenvironment to expand and refill the peripheral blood cell pool after ablation. Roundabout guidance receptor 4 (Robo4) is a transmembrane protein exclusive to endothelial cells and is vital in preserving vascular integrity. Hence, the hypothesis is that Robo4 maintains the integrity of bone marrow endothelial cells following radiotherapy. We created an endothelial cell injury model with γ-radiation before Robo4 gene manipulation using lentiviral-mediated RNAi and gene overexpression techniques. We demonstrate that Robo4 and specific mesenchymal proteins (Fibronectin, Vimentin, αSma, and S100A4) are upregulated in endothelial cells exposed to irradiation (IR). We found that Robo4 depletion increases the expression of endoglin (CD105), an auxiliary receptor for the transforming growth factor (TGF-ß) family of proteins, and promotes endothelial-to-mesenchymal transition (End-MT) through activation of both the canonical (Smad) and non-canonical (AKT/NF-κB) signaling pathways to facilitate Snail1 activation and its nuclear translocation. Endothelial Robo4 overexpression stimulates the expression of immunoglobulin-like adhesion molecules (ICAM-1 and VCAM-1) and alleviates irradiation-induced End-MT. Our coculture model showed that transcriptional downregulation of endothelial Robo4 reduces HSPC proliferation and increases HSC quiescence and apoptosis. However, Robo4 overexpression mitigated the damaged endothelium's suppressive effects on HSC proliferation and differentiation. These findings indicate that by controlling End-MT, Robo4 preserves microvascular integrity after radiation preconditioning, protects endothelial function, and lessens the inhibitory effect of damaged endothelium on hematopoietic reconstitution.

Celastrol inhibits platelet function and thrombus formation.

INTRODUCTION: Celastrol is an active pentacyclic triterpenoid extracted from Tripterygium wilfordii and has anti-inflammatory and anti-tumor properties. Whether Celastrol modulates platelet function remains unknown. Our study investigated its role in platelet function and thrombosis. METHODS: Human platelets were isolated and incubated with Celastrol (0, 1, 3 and 5 µM) at 37 °C for 1 h to measure platelet aggregation, granules release, spreading, thrombin-induced clot retraction and intracellular calcium mobilization. Additionally, Celastrol (2 mg/kg) was intraperitoneally administrated into mice to evaluate hemostasis and thrombosis in vivo. RESULTS: Celastrol treatment significantly decreased platelet aggregation and secretion of dense or alpha granules induced by collagen-related peptide (CRP) or thrombin in a dose-dependent manner. Additionally, Celastrol-treated platelets showed a dramatically reduced spreading activity and decreased clot retraction. Moreover, Celastrol administration prolonged tail bleeding time and inhibited formation of arterial/venous thrombosis. Furthermore, Celastrol significantly reduced calcium mobilization. CONCLUSION: Celastrol inhibits platelet function and venous/arterial thrombosis, implying that it might be utilized for treating thrombotic diseases.

Platelet-Derived TGF-ß1 Promotes Deep Vein Thrombosis.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) modulates multiple cellular functions during development and tissue homeostasis. A large amount of TGF-ß1 is stored in platelet α-granules and released upon platelet activation. Whether platelet-derived TGF-ß1 plays a role in venous thrombosis remains unclear. This study intends to assess the role of platelet-derived TGF-ß1 in the development of venous thrombosis in mice. MATERIAL AND METHODS: TGF-ß1flox/flox and platelet-specific TGF-ß1-/- mice were utilized to assess platelet function in vitro, arterial thrombosis induced by FeCl3, tail bleeding time, prothrombin time (PT), activated partial thromboplastin time (APTT), and deep vein thrombosis induced through ligation of the inferior vena cava (IVC). The IVC sample was collected to measure accumulation of neutrophils, monocytes, and the formation of neutrophil extracellular traps (NETs) by immunofluorescence staining. RESULTS: TGF-ß1 deficiency in platelets did not affect the number of circulating platelets, platelet aggregation, adenosine triphosphate release, and integrin αIIbß3 activation. Meanwhile, TGF-ß1 deficiency did not alter the arterial thrombus formation, hemostasis, and coagulation time (PT and APTT), but significantly impaired venous thrombus formation, inhibited the recruitment and accumulation of neutrophils and monocytes in thrombi, as well as reduced formation of NETs and platelet-neutrophil complex. In addition, adoptive transfer of TGF-ß1flox/flox platelets to TGF-ß1-/- mice rescued the impaired venous thrombus formation, recruitment of leukocytes and monocytes, as well as the NETs formation. CONCLUSION: In conclusion, platelet-derived TGF-ß1 positively modulates venous thrombus formation in mice, indicating that targeting TGF-ß1 might be a novel approach for treating venous thrombosis without increasing the risk of bleeding.

Long-term analysis of cellular immunity in patients with RRMM treated with CAR-T cell therapy.

Chimeric antigen receptor T (CAR-T) cell therapy exhibits remarkable efficacy against refractory or relapsed multiple myeloma (RRMM); however, the immune deficiency following CAR-Ts infusion has not been well studied. In this study, 126 patients who achieved remission post-CAR-Ts infusion were evaluated for cellular immunity. Following lymphodepletion (LD) chemotherapy, the absolute lymphocyte count (ALC) and absolute counts of lymphocyte subsets were significantly lower than baseline at D0. Grade ≥ 3 lymphopenia occurred in 99% of patients within the first 30 days, with most being resolved by 180 days. The median CD4+ T-cell count was consistently below baseline and the lower limit of normal (LLN) levels at follow-up. Conversely, the median CD8+ T-cell count returned to the baseline and LLN levels by D30. The median B-cell count remained lower than baseline level at D60 and returned to baseline and LLN levels at D180. In the first 30 days, 27 (21.4%) patients had 29 infections, with the majority being mild to moderate in severity (21/29; 72.4%). After day 30, 44 (34.9%) patients had 56 infections, including 20 severe infections. One patient died from bacteremia at 3.8 months post-CAR-Ts infusion. In conclusion, most patients with RRMM experienced cellular immune deficiency caused by LD chemotherapy and CAR-Ts infusion. The ALC and most lymphocyte subsets gradually recovered after day 30 of CAR-Ts infusion, except for CD4+ T cells. Some patients experience prolonged CD4+ T-cell immunosuppression without severe infection.

MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis.

Human MutT Homolog 1 (MTH1) is a nucleotide pool sanitization enzyme that hydrolyzes oxidized nucleotides to prevent their mis-incorporation into DNA under oxidative stress. Expression and functional roles of MTH1 in platelets are not known. Here, we show MTH1 expression in platelets and its deficiency impairs hemostasis and arterial/venous thrombosis in vivo. MTH1 deficiency reduced platelet aggregation, phosphatidylserine exposure and calcium mobilization induced by thrombin but not by collagen-related peptide (CRP) along with decreased mitochondrial ATP production. Thrombin but not CRP induced Ca2+-dependent mitochondria reactive oxygen species generation. Mechanistically, MTH1 deficiency caused mitochondrial DNA oxidative damage and reduced the expression of cytochrome c oxidase 1. Furthermore, MTH1 exerts a similar role in human platelet function. Our study suggests that MTH1 exerts a protective function against oxidative stress in platelets and indicates that MTH1 could be a potential therapeutic target for the prevention of thrombotic diseases.

A combination of pre-infusion serum ferritin, CRP and IL-6 predicts outcome in relapsed/refractory multiple myeloma patients treated with CAR-T cells.

Background: Chimeric antigen receptor - T (CAR-T) cell therapy has shown remarkable efficacy in patients with relapsed/refractory multiple myeloma (R/R MM). However, a subset of patients still experienced progression or relapse, and the predictors of prognosis are little known. We analyzed the inflammatory markers before CAR-T cell infusion, to clarify their correlation with survival and toxicity. Methods: This study involved 109 R/R MM patients who received CAR-T therapy between June 2017 and July 2021. Inflammatory markers, including ferritin, c-reactive protein (CRP), and interleukin-6 (IL-6) before CAR-T cell infusion were detected and then categorized by quartiles. Adverse events and clinical outcomes were compared between patients with upper quartile of inflammatory markers and patients with lower three quartiles of inflammatory markers. An inflammatory prognostic index (InPI) based on these three inflammatory markers was developed in this study. Patients were divided into 3 groups according to the InPI score, progression-free survival (PFS) and overall survival (OS) were compared among the groups. In addition, we explored the correlation between cytokine release syndrome (CRS) and pre-infusion inflammatory markers. Results: We found that the pre-infusion high ferritin (hazard ratio [HR], 3.382; 95% confidence interval [CI], 1.667 to 6.863; P = .0007), high CRP (HR, 2.043; 95% CI, 1.019 to 4.097; P = .044), and high IL-6 (HR, 3.298; 95% CI, 1.598 to 6.808; P = .0013) were significantly associated with inferior OS. The formula of the InPI score was based on the HR value of these 3 variables. Three risk groups were formed: (good, 0 to 0.5 point; intermediate, 1 to 1.5 points; poor, 2 to 2.5 points). Median OS for patients with good, intermediate, and poor InPI was not reached, 24 months, and 4 months, respectively, and median PFS was 19.1 months, 12.3 months, and 2.9 months, respectively. In the cox proportional hazards model, poor InPI remained an independent prognostic factor for PFS and OS. Pre-infusion ferritin was negatively associated with CAR T-cell expansion normalized to baseline tumor burden. Spearman correlation analysis showed that pre-infusion ferritin and IL-6 levels positively correlated with the grade of CRS (P = .0369 and P = .0117, respectively). The incidence of severe CRS was higher in patients with high IL-6 compared with patients with low IL-6 (26% vs. 9%, P = .0405). Pre-infusion ferritin, CRP and IL-6 were positively correlated with each peak values within the first month after infusion. Conclusions: Our results suggest that patients with elevated inflammation markers before CAR-T cell infusion are more likely to have poor prognosis.

Dimethyl fumarate possesses antiplatelet and antithrombotic properties.

BACKGROUND: Dimethyl fumarate (DMF) is a methyl ester of fumaric acid and has been approved for treating multiple sclerosis (MS) and psoriasis due to anti-inflammatory effect. There is a close association between platelets and the pathogenesis of MS. Whether DMF affects platelet function remains unclear. Our study intends to evaluate DMF's effect on platelet function. METHODS: Washed human platelets were treated with different concentrations of DMF (0, 50, 100 and 200 µM) at 37 °C for 1 h followed by analysis of platelet aggregation, granules release, receptors expression, spreading and clot retraction. In addition, mice received intraperitoneal injection of DMF (15 mg/kg) to assess tail bleeding time, arterial and venous thrombosis. RESULTS: DMF significantly inhibited platelet aggregation and the release of dense/alpha granules in response to collagen-related peptide (CRP) or thrombin stimulation dose-dependently without altering the expression of platelet receptors αIIbß3, GPIbα, and GPVI. In addition, DMF-treated platelets presented significantly reduced spreading on collagen or fibrinogen and thrombin-mediated clot retraction along with the decreased phosphorylation of c-Src and PLCγ2. Moreover, administration of DMF into mice significantly prolonged the tail bleeding time and impaired arterial and venous thrombus formation. Furthermore, DMF reduced the generation of intracellular reactive oxygen species and calcium mobilization, and inhibited NF-κB activation and the phosphorylation of ERK1/2, p38 and AKT. CONCLUSION: DMF inhibits platelet function and arterial/venous thrombus formation. Considering the presence of thrombotic events in MS, our study indicates that DMF treatment for patients with MS might obtain both anti-inflammatory and anti-thrombotic benefits.

Anti-G Protein-Coupled Receptor, Class C Group 5 Member D Chimeric Antigen Receptor T Cells in Patients With Relapsed or Refractory Multiple Myeloma: A Single-Arm, Phase â ¡ Trial.

PURPOSE: G protein-coupled receptor, class C group 5 member D (GPRC5D) is considered to be a promising surface target for multiple myeloma (MM) immunotherapy. Here, we report the efficacy and safety of anti-GPRC5D chimeric antigen receptor (CAR) T cells in patients with relapsed or refractory (R/R) MM. METHODS: This phase Ⅱ, single-arm study enrolled patients (18-70 years) with R/R MM. Lymphodepletion was performed before patients received 2 × 106/kg anti-GPRC5D CAR T cells. The primary end point was the proportion of patients who achieved an overall response. Safety was also evaluated in eligible patients. RESULTS: From September 1, 2021, to March 23, 2022, 33 patients were infused with anti-GPRC5D CAR T cells. At a median follow-up of 5.2 months (range, 3.2-8.9), the overall response rate was 91% (95% CI, 76 to 98; 30 of 33 patients), including 11 (33%) stringent complete responses, 10 (30%) complete responses, four (12%) very good partial responses, and five (15%) partial responses. Partial responses or better were observed in nine (100%) of nine patients with previous anti-B-cell maturation antigen (BCMA) CAR T-cell therapy, including two patients who had received repeated anti-BCMA CAR T-cell infusions with no responses at the last time. Grade 3 or higher hematologic toxicities were neutropenia (33 [100%]), anemia (17 [52%]), and thrombocytopenia (15 [45%]). Cytokine release syndrome occurred in 25 (76%) of 33 patients (all were grade 1 or 2), and neurotoxicities in three patients (one grade 2 and one grade 3 ICANSs and one grade 3 headache). CONCLUSION: Anti-GPRC5D CAR T-cell therapy showed an encouraging clinical efficacy and manageable safety profile in patients with R/R MM. For patients with MM that progressed after anti-BCMA CAR T-cell therapy or that is refractory to anti-BCMA CAR T cell, anti-GPRC5D CAR T-cell therapy might be a potential alternative option.

Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions.

BACKGROUND: Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable to overcome the preconditioning damages. This study aims to ascertain the role of Roundabout 4 (Robo4) in regulating irradiation-induced damage to the endothelium. METHODS: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. Robo4 expression in the endothelial cells was manipulated employing lentiviral-mediated RNAi and gene overexpression technology before irradiation treatment. The permeability of endothelial cells was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules, adherens junctions, tight junctions, and gap junctions. Using Transwell endothelial monolayer staining, FITC-Dextran permeability, and gap junction-mediated intercellular communication (GJIC) assays, we determined the changes in endothelial functions after Robo4 gene manipulation and irradiation. Moreover, we measured the proportion of CD31 expression in endothelial cells by flow cytometry. We analyzed variations between two or multiple groups using Student's t-tests and ANOVA. RESULTS: Ionizing radiation upregulates Robo4 expression but disrupts endothelial junctional molecules. Robo4 deletion causes further degradation of endothelial junctions hence increasing the permeability of the endothelial cell monolayer. Robo4 knockdown in microvascular endothelial cells increases the degradation and delocalization of ZO-1, PECAM-1, occludin, and claudin-5 molecules after irradiation. Conversely, connexin 43 expression increases after silencing Robo4 in endothelial cells to induce permeability but are readily destroyed when exposed to 10 Gy of gamma radiation. Also, Robo4 knockdown enhances Y731-VE-cadherin phosphorylation leading to the depletion and destabilization of VE-cadherin at the endothelial junctions following irradiation. However, Robo4 overexpression mitigates irradiation-induced degradation of tight junctional proteins and stabilizes claudin-5 and ZO-1 distribution. Finally, the enhanced expression of Robo4 ameliorates the irradiation-induced depletion of VE-cadherin and connexin 43, improves the integrity of microvascular endothelial cell junctions, and decreases permeability. CONCLUSION: This study reveals that Robo4 maintains microvascular integrity after radiation preconditioning treatment by regulating endothelial permeability and protecting endothelial functions. Our results also provided a potential mechanism to repair the bone marrow vascular niche after irradiation by modulating Robo4 expression.

Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice.

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from wild type or PDE5A-/- mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (ROS) generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK, and AKT. FeCl3-induced arterial thrombosis and venous thrombosis were assessed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A-deficient mice presented significantly prolonged tail bleeding time and delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression, and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production, and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK, and AKT. In conclusion, PDE5A modulates platelet activation and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

Asebogenin suppresses thrombus formation via inhibition of Syk phosphorylation.

BACKGROUND AND PURPOSE: Thrombosis is a major cause of morbidity and mortality worldwide. Platelet activation by exposed collagen through glycoprotein VI (GPVI) and formation of neutrophil extracellular traps (NETs) are critical pathogenic factors for arterial and venous thrombosis. Both events are regulated by spleen tyrosine kinase (Syk)-mediated signalling events. Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown. This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms. EXPERIMENTAL APPROACH: Platelet aggregation was assessed using an aggregometer. Platelet P-selectin exposure, integrin activation and calcium mobilization were determined by flow cytometry. NETs formation was assessed by SYTOX Green staining and immunohistochemistry. Quantitative phosphoproteomics, microscale thermophoresis, in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin. The in vivo effects of asebogenin on arterial thrombosis were investigated using FeCl3 -induced and laser-induced injury models, whereas those of venous thrombosis were induced by stenosis of the inferior vena cava. KEY RESULTS: Asebogenin inhibited a series of GPVI-induced platelet responses and suppressed NETs formation induced by proinflammatory stimuli. Mechanistically, asebogenin directly interfered with the phosphorylation of Syk at Tyr525/526, which is important for its activation. Further, asebogenin suppressed arterial thrombosis demonstrated by decreased platelet accumulation and fibrin generation and attenuated venous thrombosis determined by reduced neutrophil accumulation and NETs formation, without increasing bleeding risk. CONCLUSION AND IMPLICATIONS: Asebogenin exhibits potent antithrombotic effects by targeting Syk and is a potential lead compound for the development of efficient and safe antithrombotic agents.

Prolonged hematological toxicity in patients receiving BCMA/CD19 CAR-T-cell therapy for relapsed or refractory multiple myeloma.

Although chimeric antigen receptor T (CAR-T) cell therapy has been indicated to be effective in treating relapsed or refractory multiple myeloma (R/R MM), severe hematological toxicity (HT) remains an intractable issue. This study enrolled 54 patients with R/R MM following combined infusion of anti-CD19 and anti-BCMA CAR-T cells. The results showed that the rates of severe cytopenia were high, including severe neutropenia (28/54, 52%), severe anemia (15/54, 28%), and severe thrombocytopenia (18/54, 33%). Moreover, the incidence of prolonged HT (PHT) on Day 28 post-infusion was 52% (28/54), including 46% for severe neutropenia, 30% for severe anemia, and 31% for severe thrombocytopenia. Patients with PHT had a poorer median progression-free survival (PFS) and overall survival (OS) than patients without PHT (P=0.011; P=0.007). Furthermore, Cox regression analyses showed that PHT was an independent risk factor for PFS and OS. Univariate analyses showed that IFNγ (OR: 1.046; 95% CI: 1.002-1.093, P=0.042) and severe HT after lymphodepletion chemotherapy (OR: 0.082; 95% CI: 0.017-0.404; P=0.002) were independent risk factors for PHT. In conclusion, these results indicated that PHT was associated with poor outcomes following CAR-T-cell therapy in MM patients. Early detection and management of PHT would be beneficial for the prevention of life-threatening complications and improvement in the survival of patients after CAR-T-cell therapy. Clinical trial registration: This trial was registered on 1 May 2017 at http://www.chictr.org.cn as ChiCTR-OIC-17011272.

Efficacy and safety of CD19-specific CAR-T cell-based therapy in secondary central nervous system lymphoma.

Encouraging response has been achieved in relapsed/refractory (R/R) B-cell lymphoma treated by chimeric antigen receptor T (CAR-T) cells. The efficacy and safety of CAR-T cells in central nervous system lymphoma (CNSL) are still elusive. Here, we retrospectively analyzed 15 patients with R/R secondary CNSL receiving CD19-specific CAR-T cell-based therapy. The patients were infused with CD19, CD19/CD20 or CD19/CD22 CAR-T cells following a conditioning regimen of cyclophosphamide and fludarabine. The overall response rate was 73.3% (11/15), including 9 (60%) with complete remission (CR) and 2 (13.3%) with partial remission (PR). During a median follow-up of 12 months, the median progression-free survival (PFS) was 4 months, and the median overall survival (OS) was 9 months. Of 12 patients with systemic tumor infiltration, 7 (58.3%) achieved CR in CNS, and 5 (41.7%) achieved CR both systemically and in CNS. Median DOR for CNS and systemic disease were 8 and 4 months, respectively. At the end point of observation, of the 7 patients achieved CNS disease CR, one was still alive with sustained CR of CNS disease and systemic disease. The other 6 died of systemic progression. Of the 15 patients, 11 (73.3%) experienced grades 1-2 CRS, and no patient had grades 3-4 CRS. Immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 3 (20%) patients, including 1 (6.6%) with grade 4 ICANS. All the CRS or ICANS were manageable. The CD19-specific CAR-T cell-based therapy appeared to be a promising therapeutic approach in secondary CNSL, based on its antitumor effects and an acceptable side effect profile, meanwhile more strategies are needed to maintain the response.

Liposomes trigger bone marrow niche macrophage "foam" cell formation and affect hematopoiesis in mice.

Liposomes are the most widely used nanocarrier platform for the delivery of therapeutic and diagnostic agents, and a number of liposomes have been approved for use in clinical practice. After systemic administration, most liposomes are cleared by macrophages in the mononuclear phagocyte system, such as the liver and bone marrow (BM). However, the majority of studies have focused on investigating the therapeutic results of liposomal drugs, and too few studies have evaluated the potential side effects of empty nanocarriers on the functions of macrophages in the mononuclear phagocyte system. Here, we evaluate the potential effects of empty liposomes on the functions of BM niche macrophages. Following liposome administration, we observed lipid droplet (LD) accumulation in cultured primary macrophages and BM niche macrophages. We found that these LD-accumulating macrophages, similar to foam cells, exhibited increased expression of inflammatory cytokines, such as IL-1ß and IL-6. We further provided evidence that liposome deposition and degradation induced LD biogenesis on the endoplasmic reticulum membrane and subsequently disturbed endoplasmic reticulum homeostasis and activated the inositol-requiring transmembrane kinase/endoribonuclease 1α/NF-κB signaling pathway, which is responsible for the inflammatory activation of macrophages after liposome engulfment. Finally, we also showed the side effects of dysfunctional BM niche macrophages on hematopoiesis in mice, such as the promotion of myeloid-biased output and impairment of erythropoiesis. This study not only draws attention to the safety of liposomal drugs in clinical practice but also provides new directions for the design of lipid-based drug carriers in preclinical studies.

Targeting cyclin-dependent kinases 4/6 inhibits survival of megakaryoblasts in acute megakaryoblastic leukaemia.

Acute megakaryoblastic leukaemia (AMKL) is characterized by expansion of megakaryoblasts, which are hyper-proliferative cells that fail to undergo differentiation. Insight to the cell-cycle regulation revealed important events in early or late megakaryocytes (MKs) maturation; the cyclin-dependent kinases 4 and 6 (CDK4/6) have been reported to participate in the development of progenitor megakaryocytes, mainly by promoting cell cycle progression and DNA polyploidization. However, it remains unclear whether the continuous proliferation, but not differentiation, of megakaryoblasts is related to an aberrant regulation of CDK4/6 in AMKL. Here, we found that CDK4/6 were up regulated in patients with AMKL, and persistently maintained at a high level during the differentiation of abnormal megakaryocytes in vitro, according to a database and western blot. Additionally, AMKL cells were exceptionally reliant on the cell cycle regulators CDK4 or 6, as blocking their activity using an inhibitor or short hairpin RNA (shRNA) significantly reduced the proliferation of 6133/MPL megakaryocytes, reduced DNA polyploidy, induced apoptosis, decreased the level of phosphorylated retinoblastoma protein (p-Rb), and activation of caspase 3. Additionally, CDK4/6 inhibitors and shRNA reduced the numbers of leukemia cells in the liver and bone marrow (BM), alleviated hepatosplenomegaly, and prolonged the survival of AMKL-transplanted mice. These results suggested that blocking the activity of CDK4/6 may represent an effective approach to control megakaryoblasts in AMKL.