Development of a clinical prognostic model for primary plasma cell leukemia patients treated with novel agents: a multicenter retrospective cohort study.

The prognosis of primary plasma cell leukemia (pPCL) is poor, and the relevant prognostic factors are incompletely understood. We aimed to explore the prognostic factors and develop a validated prognostic prediction model for pPCL patients in the new era. This multicenter retrospective study was conducted across 16 hospitals in China. Cox proportional hazards regression analysis was used to develop a prediction model. The predictive performance of the model was assessed using multiple metrics. Internal validation was conducted using bootstrap resampling. A total of 102 pPCL patients were included in this study, and 57 (55.9%) were male. The 12-month, 24-month, and 36-month OS rates for pPCL patients were 75.4%, 58.3%, and 47.6%, respectively. An overall survival prognostic nomogram for pPCL patients was established by integrating independent prognostic factors, including age, B2MG, and del17p. The nomogram exhibited good performance, with a C-index of 0.720 (95% CI 0.642-0.797) and an AUC of 0.653. Bootstrap validation yielded a C-index of 0.721 (95% CI 0.629-0.787) and an AUC of 0.653 (95% CI 0.546-0.759), indicating a relatively good fit of the calibration curve. A nomogram incorporating age, B2MG grade, and del17p were developed and validated to accurately and consistently predict the prognosis of pPCL patients.

GSNOR overexpression enhances CAR-T cell stemness and anti-tumor function by enforcing mitochondrial fitness.

Chimeric antigen receptor-T (CAR-T) cell has been developed as a promising agent for patients with refractory or relapsed lymphoma and leukemia, but not all the recipients could achieve a long-lasting remission. The limited capacity of in vivo expansion and memory differentiation post activation is one of the major reasons for suboptimal CAR-T therapeutic efficiency. Nitric oxide (NO) plays multifaceted roles in mitochondrial dynamics and T cell activation, but its function on CAR-T cell persistence and anti-tumor efficacy remains unknown. Herein, we found the continuous signaling from CAR not only promotes excessive NO production, but also suppressed S-nitrosoglutathione reductase (GSNOR) expression in T cells, which collectively led to increased protein S-nitrosylation, resulting in impaired mitochondrial fitness and deficiency of T cell stemness. Intriguingly, enforced expression of GSNOR promoted memory differentiation of CAR-T cell after immune activation, rendered CAR-T better resistance to mitochondrial dysfunction, further enhanced CAR-T cell expansion and anti-tumor capacity in vitro and in a mouse tumor model. Thus, we revealed a critical role of NO in restricting CAR-T cell persistence and functionality, and defined that GSNOR overexpression may provide a solution to combat NO stress and render patients with more durable protection from CAR-T therapy.

System-level biological effects of extremely low-frequency electromagnetic fields: an in vivo experimental review.

During the past decades, the potential effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on human health have gained great interest all around the world. Though the International Commission on Non-Ionizing Radiation Protection recommended a 100 µT, and then a 200 µT magnetic field limit, the long-term effects of ELF-EMFs on organisms and systems need to be further investigated. It was reported that both electrotherapy and possible effects on human health could be induced under ELF-EM radiation with varied EM frequencies and fields. This present article intends to systematically review the in vivo experimental outcome and the corresponding mechanisms to shed some light on the safety considerations of ELF-EMFs. This will further advance the subsequent application of electrotherapy in human health.

[Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells].

OBJECTIVE: To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. METHODS: Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. RESULTS: After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). CONCLUSION: The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Myeloid leukemia factor 1: A "double-edged sword" in health and disease.

The occurrence and development of malignancies are closely related to abnormal cell cycle regulation. Myeloid leukemia factor 1 (MLF1) is a small nucleocytoplasmic shuttling protein associated with cell cycle exit, apoptosis, and certain immune functions. Therefore, it is pertinent to explore the role of MLF1 in health and diseases. Studies to date have suggested that MLF1 could act as a double-edged sword, regulating biochemical activities directly or indirectly. In hematopoietic cells, it serves as a protective factor for the development of lineages, and in malignancies, it serves as an oncogenesis factor. The diversity of its functions depends on the binding partners, including tumor inhibitors, scaffolding molecules, mitochondrial membrane proteins, and transcription factors. Emerging evidence indicates that MLF1 influences immune responses as well. This paper reviews the structure, biological function, and research progress on MLF1 in health and diseases to provide new insights for future research.

Relapsed/refractory diffuse large B cell lymphoma with cardiac involvement: A case report and literature review.

Background: Hematological malignancies of the heart (CHMs) are extremely rare, and include leukemia, lymphoma infiltration, and multiple myeloma with extramedullary manifestations. Cardiac lymphoma can be divided into primary cardiac lymphoma (PCL) and secondary cardiac lymphoma (SCL). Compared to PCL, SCL is relatively more common. Histologically, the most frequent SCL is diffuse large B-cell lymphoma (DLBCL). The prognosis of lymphoma in patients with cardiac involvement is extremely poor. CAR T-cell immunotherapy has been recently become a highly effective treatment for relapsed or refractory diffuse large B-cell lymphoma. To date, there are no guidelines that provide a clear consensus on the management of patients with secondary heart or pericardial involvement. We report a case of relapsed/refractory DLBCL that secondarily affected the heart. Case presentation: A male patient was diagnosed with double-expressor DLBCL based on biopsies of mediastinal and peripancreatic masses and fluorescence in situ hybridization. The patient received first-line chemotherapy and anti-CD19 CAR T cell immunotherapy, but developed heart metastases after 12 months. Considering his physical condition and economic situation of the patient, two cycles of multiline chemotherapies were administered, followed by CAR-NK cell immunotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT) at another hospital. After achieving a six-month survival, the patient died of severe pneumonia. Conclusion: The response of our patient emphasizes the importance of early diagnosis and timely treatment to improve the prognosis of SCL and serves as an important reference for SCL treatment strategies.

Loss of Lkb1 in CD11c+ myeloid cells protects mice from diet-induced obesity while enhancing glucose intolerance and IL-17/IFN-γ imbalance.

Adipose tissue CD11c+ myeloid cell is an independent risk factor associated with obesity and metabolic disorders. However, the underlying molecular basis remains elusive. Here, we demonstrated that liver kinase B1 (Lkb1), a key bioenergetic sensor, is involved in CD11c+ cell-mediated immune responses in diet-induced obesity. Loss of Lkb1 in CD11c+ cells results in obesity resistance but lower glucose tolerance, which accompanies tissue-specific immune abnormalities. The accumulation and CD80's expression of Lkb1 deficient adipose-tissue specific dendritic cells but not macrophages is restrained. Additionally, the balance of IL-17A and IFN-γ remarkably tips towards the latter in fat T cells and CD11c- macrophages. Mechanistically, IFN-γ promotes apoptosis of preadipocytes and inhibits their adipogenesis while IL-17A promotes the adipogenesis in vitro, which might account in part for the fat gain resistant phenotype. In summary, these findings reveal that Lkb1 is essential for fat CD11c+ dendritic cells responding to HFD exposure and provides new insights into the IL-17A/IFN-γ balance in HFD-induced obesity.

Systematic comparation of the biological and transcriptomic landscapes of human amniotic mesenchymal stem cells under serum-containing and serum-free conditions.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are splendid cell sources for clinical application in the administration of numerous refractory and relapse diseases. Despite the preferable prospect of serum-free (SF) condition for cell product standardization and pathogenic contamination remission, yet the systematic and detailed impact upon hAMSCs at both cellular and transcriptomic levels is largely obscure. METHODS: For the purpose, we preconditioned hAMSCs under serum-containing (SC) and SF medium for 48 h and compared the biological signatures and biofunctions from the view of cell morphology, immunophenotypes, multi-lineage differentiation in vitro, cell vitality, cytokine expression, and immunosuppressive effect upon the subpopulations of T lymphocytes, together with the PI3K-AKT-mTOR signaling reactivation upon cell vitality. Meanwhile, we took advantage of RNA-SEQ and bioinformatic analyses to verify the gene expression profiling and genetic variation spectrum in the indicated hAMSCs. RESULTS: Compared with those maintained in SC medium, hAMSCs pretreated in SF conditions manifested conservation in cell morphology, immunophenotypes, adipogenic differentiation, and immunosuppressive effect upon the proliferation and activation of most of the T cell subpopulations, but with evaluated cytokine expression (e.g., TGF-ß1, IDO1, NOS2) and declined osteogenic differentiation and cell proliferation as well as proapoptotic and apoptotic cells. The declined proliferation in the SF group was efficiently rescued by PI3K-AKT-mTOR signaling reactivation. Notably, hAMSCs cultured in SF and SC conditions revealed similarities in gene expression profiling and variations in genetic mutation at the transcriptome level. Instead, based on the differentially expressed genes and variable shear event analyses, we found those genes were mainly involved in DNA synthesis-, protein metabolism-, and cell vitality-associated biological processes and signaling pathways (e.g., P53, KRAS, PI3K-Akt-mTOR). CONCLUSIONS: Collectively, our data revealed the multifaceted cellular and molecular properties of hAMSCs under SC and SF conditions, which suggested the feasibility of serum-free culture for the preferable preparation of standardized cell products for hAMSC drug development and clinical application.

Rhabdomyosarcoma of the urinary bladder in an adult: Case report and review of the literature.

Rhabdomyosarcomas with bladder involvement in adults is an extremely rare tumor with approximately 35 cases reported. Because of its rarity in adults the exact treatment modalities are not entirely clear. Treatment is based on Children's Oncology Group in RMS. There is no significant difference in stage, lymph node status, gender, and site between adults and children. The 5 year survival for pediatric RMS is 66% and for adults 22%. It is clear that our understanding and treatment of pediatric RMS is much greater than that of adult patients and the possible reason for these differences are discussed.

[Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity].

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION: DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

[Effect of Decitabine on Regulatory T Cells Relative Content in Peripheral Blood and Bone Marrow of Patients with Myelodysplastic Syndrome/Acute Myeloid Leukemia].

OBJECTIVE: To investigate regulatory T cells (Tregs) relative content in peripheral blood and bone marrow of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with or without decitabine (DAC), analyze the immunomodulatory of Tregs in pathogenesis and remission of MDS and AML, as well as effect of DAC on Tregs. METHODS: From October 2018 to February 2019, 15 patients with MDS and 49 patients with AML (newly diagnosed, treated with DAC or other chemotherapy regimens) were enrolled in this study, and 14 cases with iron deficiency or megaloblastic anemia while without malignant tumor and autoimmune disease as controls. The Tregs relative contents in bone marrow and peripheral blood were analyzed by flow cytometry, meanwhile clinical data of the objects were collected. RESULTS: In peripheral blood and bone marrow of the patients with MDS and AML, the Tregs relative contents at newly diagnosed were higher than those of the control group (P=0.05, P=0.043). The Tregs relative content of AML patients in DAC regimen treatment group was significantly lower than that in the newly diagnosed group and non-DAC chemotherapy group (P<0.05). In DAC regimen treatment group, the Tregs relative contents was significantly lower in remission group than in non-remission group (P<0.05). There was no difference between DAC regimen treatment group and control group in Tregs relative content. CONCLUSION: DAC may increase the body's anti-tumor immunity by consuming Tregs content, enhance the body's immune function to identify and kill tumor cells, thereby promote the patients' reliefs.

Bioinformatics analysis of high frequency mutations in myelodysplastic syndrome-related patients.

BACKGROUND: Myelodysplastic syndrome (MDS) is a group of hematological malignancies that may progress to acute myeloid leukemia (AML). Bioinformatics-based analysis of high-frequency mutation genes in MDS-related patients is still relatively rare, so we conducted our research to explore whether high-frequency mutation genes in MDS-related patients can play a reference role in clinical guidance and prognosis. METHODS: Next generation sequencing (NGS) technology was used to detect 32 mutations in 64 MDS-related patients. We classified the patients' genes and analyzed them by Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, protein-protein interaction (PPI) analysis, and then calculated the gene survival curve of high-frequency mutations. RESULTS: We discovered 32 mutant genes such as ASXL1, DNMT3A, KRAS, NRAS, TP53, SF3B1, and SRSF2. The overall survival (OS) of these genes decreased significantly after DNMT3A, ASXL1, RUNX1, and U2AF1 occurred mutation. These genes play a significant role in biological processes, not only in MDS but also in the occurrence and development of other diseases. Through retrospective analysis, genes associated with MDS-related diseases were identified, and their effects on the disease were predicted. CONCLUSIONS: Thirty-two mutant genes were determined in MDS and when mutations occur in DNMT3A, ASXL1, RUNX1, and U2AF1, their survival time decreases significantly. This results providing a theoretical basis for clinical and scientific research and broadening the scope of research on MDS.

Distinct BTK inhibitors differentially induce apoptosis but similarly suppress chemotaxis and lipid accumulation in mantle cell lymphoma.

BACKGROUND: The more selective second-generation BTK inhibitors (BTKi) Acalabrutinib and Zanubrutinib and the first-generation BTKi Ibrutinib are highlighted by their clinical effectiveness in mantle cell lymphoma (MCL), however, similarities and differences of their biological and molecular effects on anti-survival of MCL cells induced by these BTKi with distinct binding selectivity against BTK remain largely unknown. METHODS: AlamarBlue assays were performed to define cytotoxicity of BTKi against MCL cells, Jeko-1 and Mino. Cleaved PARP and caspase-3 levels were examined by immunoblot analysis to study BTKi-induced apoptotic effects. Biological effects of BTKi on MCL-cell chemotaxis and lipid droplet (LD) accumulation were examined in Jeko-1, Mino and primary MCL cells via Transwell and Stimulated Raman scattering imaging analysis respectively. Enzyme-linked immunoassays were used to determine CCL3 and CCL4 levels in MCL-cell culture supernatants. RNA-seq analyses identified BTKi targets which were validated by quantitative RT-PCR (qRT-PCR) and immunoblot analysis. RESULTS: Acalabrutinib and Zanubrutinib induced moderate apoptosis in Ibrutinib high-sensitive JeKo-1 cells and Ibrutinib low-sensitive Mino cells, which was accompanied by cleaved PARP and caspase-3. Such effects might be caused by the stronger ability of Ibrutinib to upregulate the expression of pro-apoptotic genes, such as HRK, GADD45A, and ATM, in JeKo-1 cells than in Mino cells, and the expression of such apoptotic genes was slightly changed by Acalabrutinib and Zanubrutinib in both JeKo-1 and Mino cells. Further, Acalabrutinib, Zanubrutinib and Ibrutinib reduced MCL-cell chemotaxis with similar efficiency, due to their similar abilities to downmodulate chemokines, such as CCL3 and CCL4. Also, these three BTKi similarly suppressed MCL-cell LD accumulation via downregulating lipogenic factors, DGAT2, SCD, ENPP2 and ACACA without significant differences. CONCLUSION: BTKi demonstrated differential capacities to induce MCL-cell apoptosis due to their distinct capabilities to regulate the expression of apoptosis-related genes, and similar biological and molecular inhibitory effects on MCL-cell chemotaxis and LD accumulation.

FOXA2 promotes prostate cancer growth in the bone.

Bone metastasis frequently occurs in advanced-stage prostate cancer (PCa) patients. Understanding the mechanisms that promote PCa-mediated bone destruction is important for the identification of therapeutic targets against this lethal disease. We found that forkhead box A2 (FOXA2) is expressed in a subset of PCa bone metastasis specimens. To determine the functional role of FOXA2 in PCa metastasis, we knocked down the expression of FOXA2 in PCa PC3 cells, which can grow in bones and elicit an osteolytic reaction. The PC3/FOXA2-knockdown cells generated fewer bone lesions following intra-tibial injection compared to control cells. Further, we found that FOXA2 knockdown decreased the expression of PTHLH, which encodes PTHrP, a well-established factor that regulates bone remodeling. These results indicate that FOXA2 is involved in PCa bone metastasis.

Development of a highly sensitive method for detection of FLT3D835Y.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. METHODS AND RESULTS: In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. CONCLUSIONS: We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

Twist1 promotes dendritic cell-mediated antitumor immunity.

Dendritic cells (DCs) play a central role in autoimmunity, immune homeostasis, and presentation of tumor antigens to T cells in order to prime antitumor responses. The number of tumor-infiltrating DCs is associated with survival and prognosis in cancer. Twist1 is a well-known regulator of tumor initiation and promotion, but whether and how DC-derived Twist1 regulates antitumor responses remains poorly understood. Here, we generated a mouse line with Twist1 conditionally depleted in DCs and found that Twist1-deficiency in DCs did not affect the DCs and T cell homeostasis under steady-state conditions; however, in melanoma models, the proportion of conventional DCs (cDCs) in draining lymph nodes (DLNs) was significantly decreased. Accordingly, a decreased ratio and number of tumor-infiltrating cDCs were observed, which reduced the recruitment of tumor-infiltrating T cells. Furthermore, production of IFN-γ, a crucial antitumor factor, by T cells, was dramatically decreased, which can further dampen the T cell antitumor functions. Collectively, our data indicate that Twist1 in DCs regulates antitumor functions by maintain the number of tumor-infiltrating DCs and T cells, and their antitumor activity.

[Effects of Human Amniotic Mesenchymal Stem Cells on Acute Graft-Versus-Host Disease in Xenotransplantation].

OBJECTIVE: To investigate the effects of human amniotic mesenchymal stem cell（AMSC） on acute graft-versus-host disease (aGVHD) in xenotransplatation. METHODS: NPG mice were injected with human PBMNC via tail vein to establish a xenografted aGVHD model. The mice in the experimental group were divided into PBMNC infusion group and PBMNC+AMSC co-infusion group, the general condition, survival time and manifestations of aGVHD were observed, the body weight and blood routine indicators were detected, the pathological changes of aGVHD target organs (lung, liver, spleen, small intestine) were observed by HE staining, and the levels of human T cells in peripheral blood, tissues and organs of mice was detected by flow cytometry. RESULTS: The manifestations of aGVHD (lassitude hunchback, shrub, weight reduction, etc.) and the pathological damage of the target organs (lung, liver, spleen, intestine) in PBMNC+AMSC co-infusion group were lighter than those in PBMNC infusion group. Moreover, the PBMNC and AMSC co-infusion significantly reduced the implantion proportion of human T lymphocytes (CD3+, CD45+) in mice and increased the ratio of CD4+/CD8+. CONCLUSION: Infusion of human-derived AMSC can attenuate the manifestations of aGVHD in mouse xenografts to a certain level, and improve the pathological damage of receptor target organs.

Lkb1 is an important regulator of Treg differentiation and proliferation of amniotic mesenchymal stem cells.

In this study, we aimed to explore the role of liver kinase b1 (Lkb1) in the biological characteristics and immune regulation of amniotic mesenchymal stem cells (AMSCs). AMSCs were identified via the cell surface markers using flow cytometry. We knocked down the expression of Lkb1 in AMSCs using lentivirus-mediated Lkb1-specific shRNA. The efficiency of the knockdown was detected by flow cytometry, RT-qPCR, and western blot. The AMSC-related phenotype was determined by flow cytometric analysis via staining surface markers. Fibroblast colony-forming cells (CFU-F) assay and Ki-67 intracellular staining assay were used to determine the proliferative capacity. The differentiated and immunosuppressive capabilities were determined by conditional induction of differentiation and co-culture experiments. We observed that AMSCs along with Lkb1 knockdown (AMSCs-Lkb1) displayed similar cellular morphology and surface antigen expression patterns as those observed in AMSCs. However, AMSCs-Lkb1 exhibited an enhanced differentiation capacity towards osteogenesis and chondrogenesis while it showed defective proliferation and increased apoptosis. Furthermore, AMSCs-Lkb1 showed an enhanced immunosuppressive capacity by directly inhibiting conventional T cells and indirectly inducing production of regulatory T cells (Treg). Interestingly, Treg produced by AMSCs-Lkb1 displayed stronger proliferative capacity as compared to those produced by AMSCs. Our results indicate that Lkb1 plays a vital role in maintaining self-renewal of AMSCs and regulating immune equivalence, and may hold potential for the clinical management of diseases such as GVHD.

Destabilization of ROR1 enhances activity of Ibrutinib against chronic lymphocytic leukemia in vivo.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an onco-embryonic antigen presented on chronic lymphocytic leukemia (CLL), but not on normal adult tissues, which promotes CLL-cell survival. Here, ROR1 was identified as a new client of Heat Shock Protein 90 (HSP90) via a mass spectrometry-based screen for ROR1-associated partners followed by co-immunoprecipitation (co-IP) analysis. A binding motif (ELHHPNIV) on ROR1 for HSP90 was revealed, which forms a αC-ß4 loop and is necessary for HSP90-facilitated ROR1 stabilization. We also found that targeting HSP90 leads to ROR1 degradation in a ubiquitin-proteasome dependent pathway, by which pro-survival ROR1 signaling was attenuated in CLL. Based on our previous finding that a humanized monoclonal antibody against ROR1 increases the activity of Ibrutinib against CLL, which is currently undergoing evaluation in clinical trials for the treatment of B-cell lymphoid malignancies, we then provided evidence that treatment with HSP90 inhibitor (17-DMAG) enhances anti-CLL activity of Ibrutinib in vitro and in vivo, by down-modulating ROR1. iTRAQ-based quantitative proteomic analysis of other HSP90 oncogenic clients in addition to ROR1, followed by GO/KEGG enrichment analysis, showed that Bruton's Tyrosine Kinase (BTK), B-lymphoid Tyrosine Kinase (BLK), Lymphocyte-specific Protein Tyrosine Kinase (LCK), or LCK/YES-Related Novel Protein Tyrosine Kinase (LYN), as HSP90 clients, were significantly involved in 11 biological processes and 6 signaling pathways. However, immunoblotting validation confirmed that Ibrutinib treatment dramatically deprived HSP90 inhibitors, 17-DMAG, AUY922 or PU-H71, of inducing the degradation of BTK, BLK, LCK or LYN, but not ROR1. Collectively, our data suggested that depletion of ROR1 induced by targeting HSP90 might facilitate the enhancement of Ibrutinib activity against CLL.