BackgroundResveratrol (RSV) has been reported to stimulate osteoblast differentiation in which Wnt/β-catenin signaling pathway played a crucial role. However, whether and how RSV activated Wnt/β-catenin pathway in osteogenic differentiation still remained elusive.MethodsIn vivo polymethylmethacrylate (PMMA) particle-induced osteolysis (PIO) mouse model and in vitro PMMA particle-stimulated mouse mesenchymal stem cells (mMSCs) experiments were established. Relative expression levels of lncRNA KCNQ1OT1, β-catenin, Runx2, Osterix and osteocalcin were determined using quantitative Real-Time PCR. Western blotting was used to measure β-catenin protein expression. In addition, the alkaline phosphatase activity and mineral deposition level using alizarin red S staining were performed to examine osteogenic differentiation status. The interaction between KCNQ1OT1 and β-catenin was confirmed by RNA pull down assay.ResultsRSV significantly attenuated PIO in vivo and PMMA-particle inhibition of osteogenic differentiation of mMSCs. Moreover, KCNQ1OT1 exerted the similar function in mMSCs by regulating β-catenin. Further study demonstrated that RSV exerted its effect on osteoblastic differentiation by regulating KCNQ1OT1. Consequently, RSV alleviated PMMA-particle inhibition of osteoblastic differentiation via Wnt/β-catenin pathway activation in vivo and in vitro.ConclusionRSV accelerated osteoblast differentiation by regulating lncRNA KCNQ1OT1 via Wnt/β-catenin pathway activation, indicating the functional role of RSV in modulating osteogenesis.
FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of our previous study that ibrutinib (9) exhibited selective and moderate inhibitory activity against FLT3-ITD positive AML cells, through a structure-guided drug design approach, we have discovered a new type II FLT3 kinase inhibitor, compound 14 (CHMFL-FLT3-213), which exhibited highly potent inhibitory effects against FLT3-ITD mutant and associated oncogenic mutations (including FLT3-D835Y/H/V, FLT3-ITD-D835Y/I/N/A/G/Del, and FLT3-ITD-F691L). In the cellular context 14 strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting cell cycle into G0/G1 phase. In the in vivo studies 14 demonstrated an acceptable bioavailability (F = 19%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (15 mg kg day, TGI = 97%) without exhibiting obvious toxicity. Compound 14 might be a potential drug candidate for FLT3-ITD positive AML.
Introduction To further improve efficacy and duration of response of CAR-T therapy for Relapsed/Refractory Multiple Myeloma (R/R MM), we have designed a dual FasT CAR-T targeting both B cell maturation antigen (BCMA), a well-established MM target, and CD19, which is expressed on MM cells and their progenitors. Here we report early results from the first-in-human multicenter clinical study (NCT04236011; NCT04182581) to determine safety, pharmacokinetics (PK) and efficacy of BCMA-CD19-directed FasT CAR-T (GC012F) in patients with R/R MM. Methods The BCMA-CD19 dual CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3z. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis, T cells were isolated and CAR-T cells were manufactured (FasT CAR platform). From September 2019 to April 2020, we enrolled 16 heavily pretreated R/R MM patients (Age range 27-71), with a median of 5 prior lines of therapies (range 2-7), 93.8% (15/16) of these patients were high risk as defined by mSMART criteria, 5 had extramedullary disease. 4 out of 16 patients had received prior anti CD38 therapy, 93.8% (15/16) patients had received prior IMiD, all patients received at least 1 prior PI and corticosteroids with 3 patients being primary refractory to last therapy. Prior to CAR-T infusion patients received a conditioning regimen over 3 days of 30 mg/m2/d fludarabine and 300 mg/m2/d cyclophosphamide. CAR-T cells were administered in a single infusion at 3 dose levels 1x105/Kg (DL1) (1 patients), 2x105/Kg (DL2) (9 patients) and 3x105/Kg (DL3) (6 patients). Results As of July 17th 2020, all 16 patients were evaluable for response assessment, 15 out of 16 patients responded to treatment (ORR 93.8%) in all dose levels with the earliest response observed at day 28. Best response to date is MRD- CR/sCR in 9/16 patients (56.3%). In DL3 100% (6/6) of patients achieved sCR, 3 at data cut off had been confirmed by PET-CT. In all response evaluable patients, 78.6% (11/14) were MRD- by flow at month 1, and 100% at month 3 (11/11) and 6 (10/10) (sensitivity by flow cytometry measured at 10-4 in 7 patients, and at 10-6 in 9 patients tested by EuroFlow with at least 1.08x107 cells analyzed). At data cut off, the median follow up time was 7.3 months, the longest follow up was 10 months post infusion. CAR-T PK in PB was monitored by qPCR and flow cytometry. The CAR-T median proliferation peak was reached on Day10 (Day8-Day14), and the median peak copy number was 140,982 (16,011-374,346) copies /ug DNA. GC012F showed an acceptable safety profile with 14 out of 16 patients experiencing a cytokine release syndrome (CRS) grade 1-2 (n=14, 87.5%) and 2 grade 3 (n=2, 12.5%). The median duration of CRS was 4 days (1-8 days). No neurotoxicity of any grade was observed. One patient (DL2) presented with fever and died shortly after Day 78 of unknown cause during the COVID-19 Pandemic. Two patients had progression of extramedullary disease while achieving MRD negativity at month 1 and 3, respectively. At landmark analysis at 6 months, all patients in DL3 had achieved and maintained MRD- sCR including patients heavily pretreated including Daratumumab - among them 83.3% (5/6) patients in DL3 had high risk features according to mSMART criteria, and 5 out of 6 patients in DL3 were assessed by 10-6 Euroflow for MRD. The study is still enrolling patients and we will continue to be monitoring safety and efficacy including duration of response. Conclusion The data of BCMA-CD19 dual FasT CAR-T showed an early and high response rate with 93.8% ORR to date with a promising early high MRD-sCR rate in the highest dose level DL3 (100%) which was sustained with a median duration of follow up of 7.3 months at cut off. The data shows very promising activity of the BCMA-CD19 dual FasT CAR-T with a favorable safety profile in R/R MM patients. 93.8% (15/16) of the treated patients exhibited high risk features - a specifically difficult to treat patient population which remains a high unmet medical need in Multiple Myeloma. This data indicates that BCMA-CD19 dual FasT CAR-T (GC012F) may present an effective new treatment option for patients with R/R MM including those with high-risk features who failed multiple prior therapies including anti-CD38. The study is still ongoing and enrolling patients, we will update the results as they become available. Disclosures Zhao: Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Chen:Gracell Biotechnologies Ltd: Current Employment. Xu:Gracell Biotechnologies Ltd: Current Employment. Zhang:Gracell Biotechnologies Ltd: Current Employment. He:Gracell Biotechnologies Co., Ltd.: Current Employment. Shi:Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Ye:Gracell Biotechnologies Co., Ltd.: Current Employment. Wang:Gracell Biotechnologies Ltd: Current Employment. Liu:Gracell Biotechnologies Co., Ltd.: Current Employment. Shen:Gracell Biotechnologies Ltd: Current Employment. Cao:Gracell Biotechnologies Ltd: Current Employment. Sersch:Gracell Biotechnologies Co., Ltd.: Current Employment.
This study aimed to investigate the mechanism of lncRNA-KCNQ1OT1 on macrophage polarization to ameliorate particle-induced osteolysis. We used polymethylmethacrylate (PMMA) to induce primary bone marrow-derived macrophages (BMMs) obtained from mice and the RAW264.7 cell line, and found that the tumor necrosis factor-alpha (TNF-α) concentration and inducible nitric oxide synthase (iNOS) expression was increased, while interleukin (IL)-10 concentration and Arg1 expression were decreased in PMMA-induced cells. KCNQ1OT1 and IL-10 expression were both suppressed and miR-21a-5p expression was promoted in PMMA-induced cells. Overexpression of KCNQ1OT1 reversed the effect of PMMA on RAW264.7 cells, such as the reduced TNF-α concentration and iNOS expression, and increased IL-10 concentration and Arg1 expression in PMMA-induced cell transfected with pcDNA-KCNQ1OT1. The luciferase assay confirmed that IL-10 is a target of miR-21a-5p. RNA immunoprecipitation (RIP) and RNA pull-down experiments demonstrated that KCNQ1OT1 functions as a miR-21a-5p decoy. Thus, lncRNA KCNQ1OT1 induces M2 macrophage polarization to ameliorate particle-induced osteolysis by inhibiting miR-21a-5p.
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