The development of chimeric antigen receptor T-cells against CD70 for renal cell carcinoma treatment.

In this study, we investigated CD70 as a promising target for renal cell carcinoma (RCC) therapy and developed a potent chimeric antigen receptor T (CAR-T) cells for potential clinical testing. CD70, found to be highly expressed in RCC tumors, was associated with decreased survival. We generated CAR-T cells expressing VHH sequence of various novel nanobodies from immunized alpaca and a single-chain variable fragment (scFv) derived from human antibody (41D12). In our in vitro experiments, anti-CD70 CAR-T cells effectively eliminated CD70-positive tumor cells while sparing CD70-negative cells. The nanobody-based CAR-T cells demonstrated significantly higher production of cytokines such as IL-2, IFN-γ and TNF-ɑ during co-culture, indicating their potential for enhanced functionality. In xenograft mouse model, these CAR-T cells exhibited remarkable anti-tumor activity, leading to the eradication of RCC tumor cells. Importantly, human T cell expansion after infusion was significantly higher in the VHH groups compared to the scFv CAR-T group. Upon re-challenging mice with RCC tumor cells, the VHH CAR-T treated group remained tumor-free, suggesting a robust and long-lasting anti-tumor response. These findings provide strong support for the potential of nanobody-based CD70 CAR-T cells as a promising therapeutic option for RCC. This warrants further development and consideration for future clinical trials and applications.

Preventive and Therapeutic Effects of a Novel JAK Inhibitor SHR0302 in Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGVHD) is one of the most common complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Janus kinase (JAK) inhibitors are considered as reliable and promising agents for patients with aGVHD. The prophylactic and therapeutic effects of SHR0302, a novel JAK inhibitor, were evaluated in aGVHD mouse models. The overall survival (OS), progression-free survival (PFS), bodyweight of mice, GVHD scores were observed and recorded. The bone marrow and spleen samples of diseased model mice or peripheral blood of patients were analyzed. SHR0302 could prevent and reverse aGVHD in mouse models with preserving graft-versus-tumor effect. Functionally, SHR0302 improved the OS and PFS, restored bodyweight, reduced GVHD scores, and reduced immune cells infiltrated in target tissues. SHR0302 treatment also enhanced the hematopoietic reconstruction compared to the control group. Mechanistically, our results suggested that SHR0302 could inhibit the activation of T cells and modulate the differentiation of helper T (Th) cells by reducing Th1 and increasing regulatory T (Treg) cells. In addition, SHR0302 decreased the expression of chemokine receptor CXCR3 on donor T cells and the secretion of cytokines or chemokines including interleukin (IL)-6, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), CXCL10, etc. thereby destroying the IFN-γ/CXCR3/CXCL10 axis which promotes the progression of GVHD. Besides, SHR0302 decreased the phosphorylation of JAK and its downstream STATs, AKT and ERK1/2, which ultimately regulated the activation, proliferation, and differentiation of lymphocytes. Experiments on primary cells from aGVHD patients also confirmed the results. In summary, our results indicated that JAK inhibitor SHR0302 might be used as a novel agent for patients with aGVHD.

[Establishment of A Mixed Chimeric Mouse Model of Allogeneic Bone Marrow Transplantation and Its Influencing Factors].

OBJECTIVE: To establish a mouse mixed chimerism (MC) model of nonmyeloablative allogeneic bone marrow transplantation(allo-BMT) and explore its affecting factors. METHODS: The MC model was established by nonmyeloablative allo-BMT followed by high-dose post-transplant cyclophosphamide (PTCY). 123 mice in the experiments was retrospectively analyzed, and the factors related with the chimerism were explored with the univariate and multivariate logistic regression analysis. A multivariate linear regression was performed by R project to obtain a mathematical model for predicting the chimeric level with relevant affecting factors. RESULTS: The model presented mixed chimerism on day 14 after transplantation, and was characterized by a donor lymphocyte infusion (DLI) which significantly promoted donor engraftment on day 15, but transfplantation of PBS in control group was failed. Among 123 mice, 47 (38.21%) mice were MC, while 76 (61.79%) mice were non-MC in 123 mice, respectively; univariate analysis showed that the baseline body weight of mice (P=0.001, 17.84±1.19 g vs 18.50±0.94 g), total body irradiation(TBI，P=0.048) and the using of cyclophosphamide (P=0.16) were affected the chimeric state of mice, while the number of infusing cells and the time of detection showed no significant effects. Multivariate regression analysis showed that under certain conditions, the body weight of mice on day 0 was an independent factor affecting chimeric levels (OR=0.493, 95% CI 0.307-0.791, P=0.003). Through R project multiple linear regression, the math model was achieved, which was chimerism=6.09-12×weight(g)+80.03×TBI(Gy)-4.4×cell-counts (× 107) +0.38×CTX (mg/kg), R2=0.5841, P<0.001. CONCLUSION: The experiment presents a method for establishing a mixed chimeric mice model after non-myeloablative bone marrow transplantation and constructs a mathematical model with relevant factors affected chimerism status.

The nuclear receptor corepressor NCoR1 regulates hematopoiesis and leukemogenesis in vivo.

Enhanced understanding of normal and malignant hematopoiesis pathways should facilitate the development of effective clinical treatment strategies for hematopoietic malignancies. Nuclear receptor corepressor 1 (NCoR1) has been implicated in transcriptional repression and embryonic organ development, but its role in hematopoiesis is yet to be fully elucidated. Here, we showed that hematopoietic-specific loss of NCoR1 leads to expansion of the hematopoietic stem cell (HSC) pool due to aberrant cell cycle entry of long-term HSCs under steady-state conditions. Moreover, NCoR1-deficient HSCs exhibited normal self-renewal capacity but severely impaired lymphoid-differentiation potential in competitive hematopoietic-reconstitution assays. Transcriptome analysis further revealed that several hematopoiesis-associated genes are regulated by NCoR1. In addition, NCoR1 deficiency in hematopoietic cells delayed the course of leukemia and promoted leukemia cell differentiation in an MLL-AF9-induced mouse model. NCoR1 and its partner, histone deacetylase 3, can modulate histone acetylation and gene transcription through binding the promoter regions of myeloid-differentiation genes. Our collective results support the critical involvement of NCoR1 in normal and malignant hematopoiesis in vivo.

MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation.

Trithorax group protein MLL5 is an important epigenetic modifier that controls cell cycle progression, chromatin architecture maintenance, and hematopoiesis. However, whether MLL5 has a role in innate antiviral immunity is largely unknown. Here we show that MLL5 suppresses the RIG-I-mediated anti-viral immune response. Mll5-deficient mice infected with vesicular stomatitis virus show enhanced anti-viral innate immunity, reduced morbidity, and viral load. Mechanistically, a fraction of MLL5 located in the cytoplasm interacts with both RIG-I and its E3 ubiquitin ligase STUB1, which promotes K48-linked polyubiquitination and proteasomal degradation of RIG-I. MLL5 deficiency attenuates the RIG-I and STUB1 association, reducing K48-linked polyubiquitination and accumulation of RIG-I protein in cells. Upon virus infection, nuclear MLL5 protein translocates from the nucleus to the cytoplasm inducing STUB1-mediated degradation of RIG-I. Our study uncovers a previously unrecognized role for MLL5 in antiviral innate immune responses and suggests a new target for controlling viral infection.

The chromatin remodeling subunit Baf200 promotes normal hematopoiesis and inhibits leukemogenesis.

BACKGROUND: Adenosine triphosphate (ATP)-dependent chromatin remodeling SWI/SNF-like BAF and PBAF complexes have been implicated in the regulation of stem cell function and cancers. Several subunits of BAF or PBAF, including BRG1, BAF53a, BAF45a, BAF180, and BAF250a, are known to be involved in hematopoiesis. Baf200, a subunit of PBAF complex, plays a pivotal role in heart morphogenesis and coronary artery angiogenesis. However, little is known on the importance of Baf200 in normal and malignant hematopoiesis. METHODS: Utilizing Tie2-Cre-, Vav-iCre-, and Mx1-Cre-mediated Baf200 gene deletion combined with fetal liver/bone marrow transplantation, we investigated the function of Baf200 in fetal and adult hematopoiesis. In addition, a mouse model of MLL-AF9-driven leukemogenesis was used to study the role of Baf200 in malignant hematopoiesis. We also explored the potential mechanism by using RNA-seq, RT-qPCR, cell cycle, and apoptosis assays. RESULTS: Tie2-Cre-mediated loss of Baf200 causes perinatal death due to defective erythropoiesis and impaired hematopoietic stem cell expansion in the fetal liver. Vav-iCre-mediated loss of Baf200 causes only mild anemia and enhanced extramedullary hematopoiesis. Fetal liver hematopoietic stem cells from Tie2-Cre + , Baf200 f/f or Vav-iCre + , Baf200 f/f embryos and bone marrow hematopoietic stem cells from Vav-iCre + , Baf200 f/f mice exhibited impaired long-term reconstitution potential in vivo. A cell-autonomous requirement of Baf200 for hematopoietic stem cell function was confirmed utilizing the interferon-inducible Mx1-Cre mouse strain. Transcriptomes analysis revealed that expression of several erythropoiesis- and hematopoiesis-associated genes were regulated by Baf200. In addition, loss of Baf200 in a mouse model of MLL-AF9-driven leukemogenesis accelerates the tumor burden and shortens the host survival. CONCLUSION: Our current studies uncover critical roles of Baf200 in both normal and malignant hematopoiesis and provide a potential therapeutic target for suppressing the progression of leukemia without interfering with normal hematopoiesis.

Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7).

Mixed lineage leukemia 5 (MLL5) protein is a trithorax family histone 3 lysine 4 (H3K4) methyltransferase that regulates diverse biological processes, including cell cycle progression, hematopoiesis and cancer. The mechanisms by which MLL5 protein stability is regulated have remained unclear to date. Here, we showed that MLL5 protein stability is cooperatively regulated by O-GlcNAc transferase (OGT) and ubiquitin-specific protease 7 (USP7). Depletion of OGT in cells led to a decrease in the MLL5 protein level through ubiquitin/proteasome-dependent proteolytic degradation, whereas ectopic expression of OGT protein suppressed MLL5 ubiquitylation. We further identified deubiquitinase USP7 as a novel MLL5-associated protein using mass spectrometry. USP7 stabilized the MLL5 protein through direct binding and deubiquitylation. Loss of USP7 induced degradation of MLL5 protein. Conversely, overexpression of USP7, but not a catalytically inactive USP7 mutant, led to decreased ubiquitylation and increased MLL5 stability. Co-immunoprecipitation and co-immunostaining assays revealed that MLL5, OGT and USP7 interact with each other to form a stable ternary complex that is predominantly located in the nucleus. In addition, upregulation of MLL5 expression was correlated with increased expression of OGT and USP7 in human primary cervical adenocarcinomas. Our results collectively reveal a novel molecular mechanism underlying regulation of MLL5 protein stability and provide new insights into the functional interplay among O-GlcNAc transferase, deubiquitinase and histone methyltransferase.

Mll2 controls cardiac lineage differentiation of mouse embryonic stem cells by promoting H3K4me3 deposition at cardiac-specific genes.

Trithorax group (TrxG) proteins play critical roles in transcriptional activation by promoting methylation of histone H3 Lysine 4 (H3K4), but the precise functions of the individual TrxG members during embryonic differentiation are not fully understood. Here we show that Mll2, a TrxG member, is required for proliferation but is dispensable for maintaining the pluripotency of mouse embryonic stem cells (ESCs). In addition, differentiation of ESCs toward mesodermal and endodermal lineages is severely altered and, in particular, the cardiac lineage differentiation of ESCs is completely abolished in the absence of Mll2. Moreover, the expression of core cardiac transcription factors and the levels of H3K4 tri-methylation of these cardiac-specific promoters are significantly decreased by the loss of Mll2. Taken together, our results reveal a critical role for Mll2 in proliferation and cardiac lineage differentiation of mouse ESCs, and provide novel molecular insight into the mechanisms of cardiac development and disease.

Antennal morphology and sensilla ultrastructure of the web-spinning sawfly Acantholyda posticalis Matsumura (Hymenoptera: Pamphiliidae).

Acantholyda posticalis (Hymenoptera: Pamphiliidae) is an important pine pest with a world-wide distribution. To clarify the olfactory receptive mechanism of A. posticalis, scanning electron microscopy and transmission electron microscopy were used to examine the morphology, ultrastructure, and distribution of antennal sensilla of adults from two sites in China. The antennae were filiform, and the flagella comprised 32-35 flagellomeres. Six sensillum types were found. Sensilla chaetica were straight setae with sharply pointed tips and without dendrites in the lumen. Sensilla trichodea were characterized by a parallel-grooved wall and one terminal pore and were innervated by four dendrites at the base. Sensilla basiconica I possessed longitudinally grooved surfaces and multiple terminal pores, with five dendrites in the lumen. Sensilla basiconica II not only had a distinct terminal pore but also had numerous tiny wall pores and many dendritic branches within the sensillum lymph. Sensilla coeloconica had deep longitudinal grooves, one terminal pore and six dendrites, while sensilla campaniformia were thick-walled with a terminal opening and sensory nerve bundles in the lumen. Sensilla chaetica and s. trichodea were most abundant and distributed over the entire antennae, while s. basiconica I and II, s. coeloconica, and s. campaniformia were restricted to the ventral flagellar surfaces. Although the shape and structure of antennae were similar in males and females, females had significantly longer antennae than males, and males had significantly more s. basiconica I than females. We compared the morphology and structure of these sensilla to other Hymenoptera and discussed their possible functions.

Mixed lineage leukemia 5 (MLL5) protein regulates cell cycle progression and E2F1-responsive gene expression via association with host cell factor-1 (HCF-1).

Trithorax group proteins methylate lysine 4 of histone 3 (H3K4) at active gene promoters. MLL5 protein, a member of the Trithorax protein family, has been implicated in the control of the cell cycle progression; however, the underlying molecular mechanism(s) have not been fully determined. In this study, we found that the MLL5 protein can associate with the cell cycle regulator "host cell factor" (HCF-1). The interaction between MLL5 and HCF-1 is mediated by the "HCF-1 binding motif" (HBM) of the MLL5 protein and the Kelch domain of the HCF-1 protein. Confocal microscopy showed that the MLL5 protein largely colocalized with HCF-1 in the nucleus. Knockdown of MLL5 resulted in reduced cell proliferation and cell cycle arrest in the G1 phase. Moreover, down-regulation of E2F1 target gene expression and decreased H3K4me3 levels at E2F1-responsive promoters were observed in MLL5 knockdown cells. Additionally, the core subunits, including ASH2L, RBBP5, and WDR5, that are necessary for effective H3K4 methyltransferase activities of the Trithorax protein complexes, were absent in the MLL5 complex, suggesting that a distinct mechanism may be used by MLL5 for exerting its H3K4 methyltransferase activity. Together, our findings demonstrate that MLL5 could associate with HCF-1 and then be recruited to E2F1-responsive promoters to stimulate H3K4 trimethylation and transcriptional activation, thereby facilitating the cell cycle G1 to S phase transition.