Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion.

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset-dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

Structure, signal transduction, activation, and inhibition of integrin αIIbß3.

Integrins are heterodimeric receptors comprising α and ß subunits. They are expressed on the cell surface and play key roles in cell adhesion, migration, and growth. Several types of integrins are expressed on the platelets, including αvß3, αIIbß3, α2ß1, α5ß1, and α6ß1. Among these, physically αIIbß3 is exclusively expressed on the platelet surface and their precursor cells, megakaryocytes. αIIbß3 adopts at least three conformations: i) bent-closed, ii) extended-closed, and iii) extended-open. The transition from conformation i) to iii) occurs when αIIbß3 is activated by stimulants. Conformation iii) possesses a high ligand affinity, which triggers integrin clustering and platelet aggregation. Platelets are indispensable for maintaining vascular system integrity and preventing bleeding. However, excessive platelet activation can result in myocardial infarction (MI) and stroke. Therefore, finding a novel strategy to stop bleeding without accelerating the risk of thrombosis is important. Regulation of αIIbß3 activation is vital for this strategy. There are a large number of molecules that facilitate or inhibit αIIbß3 activation. The interference of these molecules can accurately control the balance between hemostasis and thrombosis. This review describes the structure and signal transduction of αIIbß3, summarizes the molecules that directly or indirectly affect integrin αIIbß3 activation, and discusses some novel antiαIIbß3 drugs. This will advance our understanding of the activation of αIIbß3 and its essential role in platelet function and tumor development.

RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.

Retinoic acid (RA)-inducible gene I (RIG-I) is highly upregulated and functionally implicated in the RA-induced maturation of acute myeloid leukemia (AML) blasts. However, the underlying mechanism and the biological relevance of RIG-I expression to the maintenance of leukemogenic potential are poorly understood. Here, we show that RIG-I, without priming by foreign RNA, inhibits the Src-facilitated activation of AKT-mTOR in AML cells. Moreover, in a group of primary human AML blasts, RIG-I reduction renders the Src family kinases hyperactive in promoting AKT activation. Mechanistically, a PxxP motif in RIG-I, upon the N-terminal CARDs' association with the Src SH1 domain, competes with the AKT PxxP motif for recognizing the Src SH3 domain. In accordance, mutating PxxP motif prevents Rig-I from inhibiting AKT activation, cytokine-stimulated myeloid progenitor proliferation, and in vivo repopulating capacity of leukemia cells. Collectively, our data suggest an antileukemia activity of RIG-I via competitively inhibiting Src/AKT association.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

Integrin ß3 Deficiency Results in Hypertriglyceridemia via Disrupting LPL (Lipoprotein Lipase) Secretion.

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.

Establishment and characterization of a novel 'double-hit' follicular lymphoma cell line, FL-SJC.

About 5 per cent of follicular lymphoma (FL) cases are double-hit (DH) lymphomas. Double-hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL-SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL-SJC cells demonstrated CD19++ , CD20+ , CD22++ , HLA-DR+ , CD10+ , CD38+ , Lambda+ CD23- , CD5- and Kappa- . The chromosome karyotypic analysis confirmed the co-existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL-SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL-SJC cells, the SCID mice developed solid tumour masses within 6-8 weeks. FL-SJC cells were proven to be free of Epstein-Barr (EB) virus infection and be multidrug-resistant. In a conclusion, the FL-SJC cell line has been identified as a novel MYC/BCL2 double-hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.

PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization.

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid-induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.

Evaluation of the activity levels of rat FVIII and human FVIII delivered by adeno-associated viral vectors both in vitro and in vivo.

The development of a novel coagulation factor VIII (FVIII) expression cassette with an enhanced activity for gene therapy of hemophilia A (HA) is essential. The biological properties of several non-human FVIII sequences, such as porcine and canine, have been evaluated. Here, we compared the activity level of rat FVIII (rFVIII) and human FVIII (hFVIII) by using single-chain and dual-chain strategies in 293 T cells and the HA mice. In both in vitro and hydrodynamic injection studies, the activity of rFVIII detected by the activated partial thromboplastin time assay was higher than that of hFVIII both by single-chain (~2.96-fold and ~1.72-fold, respectively) and dual-chain (~7.69-fold and ~2.35-fold, respectively). Moreover, the dual chain exerted a potentially higher delivery efficacy compared with the single chain (~4.96-fold and ~2.99-fold, respectively). The blood loss of HA mice administrated with rFVIII was less than those with hFVIII. AAV-delivered rFVIII and hFVIII also exerted long-term therapeutic effects on HA mice and caused a transient ALT elevation. These data might help to the development of novel, optimized FVIII expression cassettes based on the amino acid difference between rFVIII and hFVIII. These data indicate that the dual-chain strategy would likely enhance the delivery efficiency of the AAV-mediated FVIII gene therapy.

Arsenic trioxide at conventional dosage does not aggravate hemorrhage in the first-line treatment of adult acute promyelocytic leukemia.

OBJECTIVES: The arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) therapy has demonstrated a tremendous success in the first-line treatment of acute promyelocytic leukemia (APL). Actually, early death (ED) is currently thought as a major challenge in APL. ATO has been reported to inhibit platelet function in vitro, and whether it increases the ED rate by exacerbating the hemorrhagic symptoms remains to be investigated. METHODS: Effects of ATO on platelet aggregation and adhesion were evaluated in vitro and in thirty-two complete remission (CR) and four newly diagnosed APL patients. Furthermore, concentrations of plasma total arsenic were monitored in APL patients via ICP-MS. RESULTS: The inhibition of platelet function, either aggregation or adhesion, did occur in vitro when the concentration of ATO reached 2 µmol/L. However, in CR APL patients receiving ATO with normal platelet count, the platelets responded normally when being activated and so did those in the newly diagnosed patients with thrombocytopenia. Our data further showed that the conventional dosage of ATO reached a plasma concentration substantially below the required concentration to inhibit platelets. CONCLUSIONS: In the first-line treatment of APL, the use of ATO is safe and effective and does not compromise the hemostatic potential that may eventually increase ED rate.

Clinical manifestations and mutation spectrum of 57 subjects with congenital factor XI deficiency in China.

Congenital factor XI (FXI) deficiency is a rare bleeding disorder with unpredictable bleeding tendency. Few studies in a large cohort have been reported regarding associations between FXI activity (FXI:C) or genotypes and bleeding symptoms currently. This study characterized clinical manifestations and mutation spectrum of 57 subjects with FXI deficiency in China. Clinical data were collected and mutations were identified by direct sequencing and determined by mRNA analysis. The result revealed bleeding symptoms were only found in 12 patients (12/57, 21.1%) with severely reduced FXI:C, and prolonged bleeding post injury/surgery as well as easy bruising were the commonest bleeding manifestations presented in respective 5 cases (5/12, 41.7%). A total number of 37 mutations were identified including 19 missense mutations, 9 nonsense mutations, 6 splice site mutations and 3 small deletions. Among them, 4 missense mutations, 5 splice mutations, 3 small deletions and a nonsense mutation were newly detected. W228*, G400V, Q263* and c.1136-4delGTTG with a total frequency of 48.3% were the most four common mutations in Chinese patients. RT-PCR analysis was carried out and confirmed that both c.596-8T>A and c.1136-4delGTTG were pathogenic due to frameshift resulting in respective truncated proteins. Our findings suggested clinical manifestations had little to do with FXI:C or genotypes, which required further study. This study, the largest investigation of FXI deficiency in China revealed that the F11 mutation spectrum of Chinese population was distinct from those of other populations earlier established.

CT characteristics in 24 patients with POEMS syndrome.

BACKGROUND: POEMS (Polyneuropathy, Organomegaly, Endocrinopathy, M protein, and Skin changes) syndrome is a complicated and rare disease. Systematic research on computed tomography (CT) imaging characteristics in POEMS syndrome is scanty. The role of CT in diagnosis needs to be assessed. PURPOSE: To retrospectively analyze the CT imaging features in 24 patients with POEMS syndrome and evaluate the role of CT in diagnosis of this disease. MATERIAL AND METHODS: Twenty-four patients with confirmed POEMS syndrome were included in the study. Chest and abdominal CT images were analyzed. RESULTS: The three minor diagnostic criteria for POEMS syndrome (extravascular volume overload, organomegaly, and bone lesions) can be detected effectively by CT. Extravascular volume overload involved multiple serous cavities: hydrothorax, hydropericardium, and ascites, which were found in 79.2%, 41.7%, and 54.2% patients, respectively. The volume of effusion was small to moderate. Organomegaly involved multiorgans: hepatomegaly was found in 45.8% patients, splenomegaly in 54.2%, and lymphadenopathy in 75% patients. Hepatospleen exhibited moderate homogeneous enlargement without local enhanced signal after injection of contrast material. Bone lesions were classified into three groups: osteosclerotic, osteolytic, and mixed lesions. Osteosclerotic lesions, taking multiple, scattered, and variably sized high-density plaque-like appearance, were found in 20.8% patients. Osteolytic lesions, exhibiting punched-out low-density image, were found in 4.2% patients. Mixed ones, holding both common characteristics of them, were detected in 8.3% patients. These CT abnormalities disappeared after effective treatment. CONCLUSION: CT plays vital role in the confirmation of the three minor diagnostic criteria for POEMS syndrome: extravascular volume overload, organomegaly, and bone lesions.

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide.

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34(+) cells, especially the LSC-enriched CD34(+)CD38(-) subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34(+) cells but not those from normal CD34(+) cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide-down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.

Eriocalyxin B ameliorates experimental autoimmune encephalomyelitis by suppressing Th1 and Th17 cells.

Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, was previously reported to have antitumor effects via multiple pathways, and these pathways are related to immune responses. In this study, we demonstrated that EriB was efficacious in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Treatment with EriB led to amelioration of EAE, which correlated with reduced spinal cord inflammation and demyelination. EriB treatment abolished encephalitogenic T-cell responses to myelin oligodendrocyte glycoprotein in an adoptive transfer EAE model. The underlying mechanism of EriB-induced effects involved inhibition of T helper (Th) 1 and Th17 cell differentiation through Janus Kinase/Signal Transducer and Activator Of Transcription and Nuclear factor-κB signaling pathways as well as elevation of reactive oxygen species. These findings indicate that EriB exerts potent antiinflammatory effects through selective modulation of pathogenic Th1 and Th17 cells by targeting critical signaling pathways. The study provides insights into the role of EriB as a unique therapeutic agent for the treatment of autoimmune diseases.

Structural framework of c-Src activation by integrin ß3.

The integrin ß3-mediated c-Src priming and activation, via the SH3 domain, is consistently associated with diseases, such as the formation of thrombosis and the migration of tumor cells. Conventionally, activation of c-Src is often induced by the binding of proline-rich sequences to its SH3 domain. Instead, integrin ß3 uses R(760)GT(762) for priming and activation. Because of the lack of structural information, it is not clear where RGT will bind to SH3, and under what mechanism this interaction can prime/activate c-Src. In this study, we present a 2.0-Å x-ray crystal structure in which SH3 is complexed with the RGT peptide. The binding site lies in the "N"-Src loop of the SH3 domain. Structure-based site-directed mutagenesis showed that perturbation on the "N"-Src loop disrupts the interaction between the SH3 domain and the RGT peptide. Furthermore, the simulated c-Src:ß3 complex based on the crystal structure of SH3:RGT suggests that the binding of the RGT peptide might disrupt the intramolecular interaction between the SH3 and linker domains, leading to the disengagement of Trp260:"C"-helix and further activation of c-Src.

HDAC6 controls the kinetics of platelet activation.

HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

Facile fabrication of redox-responsive thiol-containing drug delivery system via RAFT polymerization.

A novel kind of redox-responsive polymeric drug delivery system has been designed and prepared successfully through the coupling of the multithiol branched polymers and thiol-containing drugs. The branched poly((S-(4-vinyl) benzyl S'-propyltrithiocarbonate)-co-(poly(ethylene glycol) methacrylate)) (poly(VBPT-co-PEGMA)) was synthesized by one-pot reaction via reversible addition-fragmentation chain transfer (RAFT) copolymerization. Subsequently, the hydrophobic thiol-containing anticancer drug 6-mercaptopurine (MP) was conjugated to poly(VBPT-co-PEGMA) by thiol-disulfide exchange reaction, resulting in the formation of poly(VBPT-co-PEGMA)-S-S-MP conjugate. Due to its amphiphilicity, poly(VBPT-co-PEGMA)-S-S-MP conjugate self-assembled into amphiphilic micelles in aqueous solution. Under a reductive environment, the disassembly of polymeric micelles resulted in the MP release. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that the poly(VBPT-co-PEGMA)-S-S-MP micelles could be taken up by Raji cells (a Burkitt lymphoma cell line). The viability of the Raji cells incubated with the glutathione (GSH) mediated poly(VBPT-co-PEGMA)-S-S-MP micelles was investigated by Cell Counting Kit-8 (CCK-8) assay. The experimental results showed that the viability of the glutathione monoester (GSH-OEt) pretreated cells was lower than that without pretreatment, while the viability of the buthionine sulfoximine (BSO) pretreated cells was higher than that without pretreatment. The poly(VBPT-co-PEGMA)-S-S-MP micelles could induce the apoptosis of Raji cells, and the apoptosis behavior was dose-dependent. This redox-responsive polymer-drug conjugate provides a promising platform for the delivery of thiol-containing biological molecules.

Thrombotic events associated with immune checkpoint inhibitors and novel antithrombotic strategies to mitigate bleeding risk.

Although immunotherapy is expanding treatment options for cancer patients, the prognosis of advanced cancer remains poor, and these patients must contend with both cancers and cancer-related thrombotic events. In particular, immune checkpoint inhibitors are associated with an increased risk of atherosclerotic thrombotic events. Given the fundamental role of platelets in atherothrombosis, co-administration of antiplatelet agents is always indicated. Platelets are also involved in all steps of cancer progression. Classical antithrombotic drugs can cause inevitable hemorrhagic side effects due to blocking integrin ß3 bidirectional signaling, which regulates simultaneously thrombosis and hemostasis. Meanwhile, many promising new targets are emerging with minimal bleeding risk and desirable anti-tumor effects. This review will focus on the issue of thrombosis during immune checkpoint inhibitor treatment and the role of platelet activation in cancer progression as well as explore the mechanisms by which novel antiplatelet therapies may exert both antithrombotic and antitumor effects without excessive bleeding risk.

Acquired immune thrombotic thrombocytopenic purpura (TTP) associated with inactivated COVID-19 vaccine CoronaVac.

Corona virus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has affected the whole world. Acquired thrombotic thrombocytopenic purpura (TTP) has been reported after administration of mRNA- or adenoviral vector-based COVID-19 vaccines, including Ad26.COV2-S, BNT162b2, mRNA-1273, and ChAdOx1 nCov-19. However, whether inactivated vaccines, such as CoronaVac, could cause TTP and whether the symptoms in TTPs caused by inactivated vaccines are different from previously reported cases are unknown. In this study, two cases were reported. Both cases developed TTP after the second CoronaVac vaccination shot, but not the first. They demonstrated symptoms of fever, neurological abnormalities, renal dysfunction, thrombocytopenia, and hemolysis. Both patients achieved complete remission through several sessions of plasma exchanges and immune suppression. The incidence of TTP in Nanjing area was analyzed. The number of patients with TTP was 12 in 2019, 6 in 2020, 16 in 2021, and 19 in 2022. To the authors' knowledge, this report is the first report of TTP associated with inactivated COVID-19 vaccine (CoronaVac). The rarity and delayed onset may be due to the relatively milder immune response caused by the inactivated vaccines than mRNA-based ones. Timely plasma exchange is a vital treatment for CoronaVac-related TTP, similar to activated vaccine-related TTP.