Improved efficacy and safety of zanubrutinib versus ibrutinib in patients with relapsed/refractory chronic lymphocytic leukemia (R/R CLL) in China: a subgroup of ALPINE.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) has different epidemiology in Chinese vs. Western patients, but there are few studies of CLL/SLL in large populations of Chinese patients. ALPINE is a global phase 3 trial investigating Bruton tyrosine kinase inhibitors zanubrutinib vs. ibrutinib to treat relapsed/refractory (R/R) CLL/SLL. Here we report results from the subgroup of Chinese patients. Adults with R/R CLL/SLL were randomized 1:1 to receive zanubrutinib (160 mg twice-daily) or ibrutinib (420 mg once-daily) until disease progression or unacceptable toxicity. Endpoints included overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety. Data were analyzed descriptively. Ninety patients were randomized in China (zanubrutinib, n = 47; ibrutinib, n = 43). Baseline characteristics were balanced between groups, with fewer male patients in the zanubrutinib vs. ibrutinib group (55.3% vs. 69.8%). Median age was 60.5 years, 11% had del(17p) mutation, and 32% had tumor protein 53 (TP53) mutation. With median 25.3 months follow-up, ORR was 80.9% with zanubrutinib vs. 72.1% with ibrutinib. PFS was improved with zanubrutinib vs. ibrutinib (HR = 0.34 [95% CI, 0.15, 0.77]), and the HR for OS was 0.45 (95% CI, 0.14, 1.50). Rates of Grade ≥ 3 treatment-emergent adverse events (TEAEs; 64.4% vs. 72.1%), AEs leading to discontinuation (6.4% vs. 14.0%), and serious TEAEs (35.6% vs. 51.2%) were lower with zanubrutinib vs. ibrutinib. Zanubrutinib demonstrated improved ORR, PFS, and OS vs. ibrutinib and a more favorable safety profile in patients with R/R CLL/SLL in China. These results are consistent with the full global population of ALPINE. ClinicalTrials.gov: NCT03734016, registered November 7, 2018.

Sonidegib, a Smoothened Inhibitor, Promotes Apoptosis and Suppresses Proliferation of Natural Killer/T-Cell Lymphoma.

BACKGROUND Dysregulation of the Hedgehog (Hh) pathway modulates various aspects of hematologic and solid tumors, but its effects in human Natural killer/T-cell lymphoma (NKTCL) are unclear. Moreover, no study has examined the consequences of pharmacologically inhibiting Hh signaling in NKTCL cell lines. MATERIAL AND METHODS In this study, the expression of Smoothened (Smo) and Glioma-associated oncogene 1 (Gli1) in NKTCL tissue were scrutinized. Two human NKTCL cell lines, SNK6 and SNT8, were subjected to various doses of sonidegib (a Smo inhibitor) and incubated for distinct durations. The cell apoptosis was examined by flow cytometry, CCK-8 assay was run to assess proliferation, and protein levels were quantified by Western blotting. RESULTS Both Smo and Gli1 expression were higher in NKTCL tissue than in Lymphoid Reactive Hyperplasia (LRH). Sonidegib significantly suppressed proliferation in NKTCL cells and the effect was dose-dependent. Further analysis revealed that sonidegib treatment elevated the number of apoptotic cells in a dose- and time-dependent manner. In addition, sonidegib downregulated Smo and Gli1expression in NKTCL cells. CONCLUSIONS The Hh pathway is crucial to the development of NKTCL and thus holds huge promise as a treatment for this disease.

The dual PI3K/mTOR inhibitor NVP-BEZ235 inhibits proliferation and induces apoptosis of burkitt lymphoma cells.

BACKGROUND: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a therapy target of cancer. We aimed to confirm the effect of dual PI3K/mTOR inhibitor NVP-BEZ235 on cell proliferation and apoptosis in Burkitt lymphoma (BL) cells. METHODS: Two human BL cell lines, CA46 and RAJI were used in this study. The proliferation of BL cells was detected by manganese tricarbonyl transfer (MTT) assay. Cell cycle and apoptosis assay were examined by flow cytometric analysis. The phosphorylation levels of AKT (Thr308), AKT (Ser473), and RPS6 were evaluated by western blot analysis. RESULTS: NVP-BEZ235 significantly inhibited the proliferation of BL cells (CA46 and RAJI) and the inhibition effect was time and dose-dependent. Cell cycle analysis indicated that the cells (CA46 and RAJI) were mostly arrested in G1/G0 phase. Cell apoptosis assay showed that the late apoptotic cells were significantly increased after 72 h treatment by 100 nmol/L of NVP-BEZ235. In addition, results also found that NVP-BEZ235 reduced the phosphorylation levels of AKT (Thr308), AKT (Ser473), and PRS6 in BL cells (CA46 and RAJI). Moreover, this inhibition effect on phosphorylation was dose-dependent. CONCLUSIONS: NVP-BEZ235 effectively inhibited cell proliferation by G0/G1 cell-cycle arrest and induced apoptosis through deregulating PI3K/Akt/mTOR pathway in BL cells.

[Mechanism about LMP1 of EB Virus Promoting Plasma Blast Differentiation of DLBCL Cell via mTORC1].

OBJECTIVE: To investigate possible mechanism on protien LMP1 expressed by EBV inducing plasmablast differentiation of DLBCL cell via the mTORC1 pathway. METHODS: The expression levels of LMP1 protein, CD38 and the phosphorylation levels of p70S6K in EBV+ and EBV- DLBCL cell lines were detected by Western blot. Cell lines overexpressing LMP1 gene stablely were constructed and LMP1 gene was silenced by RNAi. The expression of LMP1 gene was verified by RT-qPCR. The expression levels of LMP1 and CD38 and the phosphorylation levels of p70S6K in each group were detected by Western blot. RESULTS: Compared with EBV-DLBCL cells, the expression of LMP1 was detected on EBV +DLBCL cells (P =0.0008), EBV +DLBCL cells had higher phosphorylation levels of p70S6K (P =0.0072) and expression levels of CD38(P =0.0091). Compared with vector group, the cells of LMP1OE group had higher expression levels of LMP1 and CD38 (P =0.0353; P <0.0001), meanwhile molecular p70S6K was phosphorylated much more(P =0.0065); expression of LMP1 mRNA was verified(P <0.0001). Compared with si-NC group, expression level of LMP1 protein(P =0.0129) was not detected and phosphorylated p70S6K disappeared of LMP1KO group (P =0.0228); meanwhile, expression of CD38 decreased,although there was no significant difference (P =0.2377). CONCLUSION: LMP1 promotes DLBCL cells plasmablast differentiation via activating mTORC1 signal pathway.

[Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line].

OBJECTIVE: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. METHODS: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. RESULTS: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. CONCLUSION: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Arsenic trioxide inhibits the proliferation of myeloma cell line through notch signaling pathway.

Arsenic Trioxide (ATO) has shown remarkable efficacy for the treatment of multiple myeloma (MM). However, the mechanism by which ATO exerts its inhibitory effect on the proliferation of myeloma cells remains to be clarified. We study the inhibitory effect of ATO at various concentrations on the proliferation of the myeloma cell line RPMI 8226 and discussed the molecular mechanism of ATO on myeloma cell line. Our results proved that ATO had a significant dose-dependent and time-dependent inhibitory effect on the expressions of the Notch receptor (Notch1) and Notch ligand (Jag2). Data from the real-time PCR assay showed that the mRNA expression levels of the Jag2 gene and its downstream gene Hes1 were both significantly down-regulated after the myeloma cells were treated with ATO while the expression of the tumor suppressor gene PTEN was up-regulated. These results elucidated the molecular mechanism underlying the ATO mediated inhibition of myeloma cell proliferation. This is the first report on the anti-myeloma activity in myeloma cells through inhibition of the Notch signaling pathway.

Comprehensive analysis of the prognostic implication and immune infiltration of CISD2 in diffuse large B-cell lymphoma.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell lymphoma in adults. CDGSH iron sulfur domain 2 (CISD2) is an iron-sulfur protein and plays a critical role of cell proliferation. The aberrant expression of CISD2 is associated with the progression of multiple cancers. However, its role in DLBCL remains unclear. Methods: The differential expression of CISD2 was identified via public databases, and quantitative real-time PCR (qRT-PCR) and western blot were used to identifed the expression of CISD2. We estimated the impact of CISD2 on clinical prognosis using the Kaplan-Meier plotter. Meanwhile, the drug sensitivity of CISD2 was assessed using CellMiner database. The 100 CISD2-related genes from STRING obtained and analyzed using the LASSO Cox regression. A CISD2 related signature for risk model (CISD2Risk) was established. The PPI network of CISD2Risk was performed, and functional enrichment was conducted through the DAVID database. The impacts of CISD2Risk on clinical features were analyzed. ESTIMATE, CIBERSORT, and MCP-counter algorithm were used to identify CISD2Risk associated with immune infiltration. Subsequently, Univariate and multivariate Cox regression analysis were applied, and a prognostic nomogram, accompanied by a calibration curve, was constructed to predict 1-, 3-, and 5-years survival probabilities. Results: CISD2 was upregulated in DLBCL patients comparing with normal controls via public datasets, similarly, CISD2 was highly expressed in DLBCL cell lines. Overexpression of CISD2 was associated with poor prognosis in DLBCL patients based on the GSE31312, the GSE32918, and GSE93984 datasets (P<0.05). Nine drugs was considered as a potential therapeutic agents for CISD2. By using the LASSO cox regression, twenty seven genes were identified to construct CISD2Risk, and biological functions of these genes might be involved in apoptosis and P53 signaling pathway. The high CISD2Risk value had a worse prognosis and therapeutic effect (P<0.05). The higher stromal score, immune score, and ESTIMATE score were associated with lowe CISD2Risk value, CISD2Risk was negatively correlated with several immune infiltrating cells (macrophages M0 and M1, CD8 T cells, CD4 naïve T cells, NK cell, etc) that might be correlated with better prognosis. Additionally, The high CISD2Risk was identified as an independent prognostic factor for DLBCL patients using both univariate and multivariate Cox regression. The nomogram produced accurate predictions and the calibration curves were in good agreement. Conclusion: Our study demonstrates that high expression of CISD2 in DLBCL patients is associated with poor prognosis. We have successfully constructed and validated a good prognostic prediction and efficacy monitoring for CISD2Risk that included 27 genes. Meanwhile, CISD2Risk may be a promising evaluator for immune infiltration and serve as a reference for clinical decision-making in DLBCL patients.

Analysis of endoplasmic reticulum stress-related gene signature for the prognosis and pattern in diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. This study aimed to determine the prognostic significance of endoplasmic reticulum (ER) stress-related genes in DLBCL. ER stress-related genes were obtained from the molecular signatures database. Gene expression data and clinical outcomes from the gene expression omnibus and TCGA datasets were collected, and differentially expressed genes (DEGs) were screened out. Gene ontology enrichment analysis, the kyoto encyclopaedia of genes and genomes pathway analysis, and geneset enrichment analysis were used to analyse the possible biological function of ER stress-related DEGs in DLBCL. Protein-protein interaction network construction using the STRING online and hub genes were identified by cytoHubba on Cytoscape software. The significant prognosis-related genes were screened, and the differential expression was validated. The immune microenvironment assessment of significant genes were evaluated. Next, the nomogram was built using univariate and multivariate Cox regression analysis. 26 ER stress-related DEGs were screened. Functional enrichment analysis showed them to be involved in the regulation of the endoplasmic reticulum mainly. NUPR1 and TRIB3 were identified as the most significant prognostic-related genes by comparison with the GSE10846, GSE11318, and TCGA datasets. NUPR1 was correlated with a good prognosis and immune infiltration in DLBCL; on the other hand, high expression of TRIB3 significantly correlated with a poor prognosis, which was an independent prognostic factor for DLBCL. In summary, we identified NUPR1 and TRIB3 as critical ER stress-related genes in DLBCL. NUPR1 might be involved in immune infiltration in DLBCL, and TRIB3 might serve as a potential therapeutic target and prognostic factor in DLBCL.

Distinguishing COVID-19 from seasonal influenza in patients under age 65 years-a retrospective observational cohort study comparing the 2009 influenza A (H1N1) and 2022 SARS-CoV-2 pandemics.

Introduction: This study explored the differences in clinical characteristics between the 2009 pandemic influenza A (H1N1) and SARS-CoV-2 BA.2 variant (Omicron) infections in patients younger than age 65 years, to improve identification of these diseases and better respond to the current epidemic. Methods: Data from 127 patients with the 2009 pandemic influenza A (H1N1) diagnosed between May and July of 2009 and 3,265 patients with Omicron diagnosed between March and May of 2022 were collected. Using a 1:2 match based on age (difference <2 years), sex, and underlying diseases, data from 115 patients with the 2009 pandemic influenza A (H1N1) infection (H1N1 group) and 230 patients with SARS-CoV-2 Omicron BA.2 infection (Omicron group) were analyzed. The clinical manifestations were compared between the groups, logistic regression was performed to identify possible independent risk factors for each group, and multiple linear regression was used to analyze the factors predicting time for nucleic acid negativization (NAN). Results: The median [interquartile range] age of the two groups was 21 [11, 26] years. Compared with the H1N1 group, the Omicron group had: lower white blood cell counts and C-reactive protein levels; less fever, nasal congestion, sore throat, cough, sputum, and headache; and more olfactory loss, muscle soreness, and lactate dehydrogenase (LDH) abnormalities. Patients in the Omicron group used fewer antibiotics and antiviral drugs, and the time for NAN was longer (17 [14,20] VS 4 [3,5] days, P<0.001). Logistic regression showed that fever, cough, headache, and increased white blood cell count were more strongly correlated with the H1N1 group, while muscle soreness and LDH abnormalities were more strongly correlated with the Omicron group. Fever (B 1.529, 95% confidence interval [0.149,2.909], P=0.030) significantly predicted a longer time for NAN in patients with Omicron. Discussion: There are significant differences in clinical characteristics between SARS-CoV-2 Omicron infection and the 2009 pandemic influenza A (H1N1) infection. Recognition of these differences has important implications for clinical practice.

The effects of vaccination on the disease severity and factors for viral clearance and hospitalization in Omicron-infected patients: A retrospective observational cohort study from recent regional outbreaks in China.

Object: This study attempted to explore the effects of vaccination on disease severity and the factors for viral clearance and hospitalization in omicron-infected patients. Methods: The clinical manifestations of 3,265 Omicron-infected patients (BA.2 lineage variant; the Omicron group) were compared with those of 226 Delta-infected patients (the Delta group). A Multi-class logistic regression model was employed to analyze the impacts of vaccination doses and intervals on disease severity; a logistic regression model to evaluate the risk factors for hospitalization; R 4.1.2 data analysis to investigate the factors for time for nucleic acid negativization (NAN). Results: Compared with the Delta group, the Omicron group reported a fast transmission, mild symptoms, and lower severity incidence, and a significant inverse correlation of vaccination dose with clinical severity (OR: 0.803, 95%CI: 0.742-0.868, p<0.001). Of the 7 or 5 categories of vaccination status, the risk of severity significantly decreased only at ≥21 days after three doses (OR: 0.618, 95% CI: 0.475-0.803, p<0.001; OR: 0.627, 95% CI: 0.482-0.815, p<0.001, respectively). The Omicron group also reported underlying illness as an independent factor for hospitalization, sore throat as a protective factor, and much shorter time for NAN [15 (12,19) vs. 16 (12,22), p<0.05]. NAN was associated positively with age, female gender, fever, cough, and disease severity, but negatively with vaccination doses. Conclusion: Booster vaccination should be advocated for COVID-19 pandemic-related control and prevention policies and adequate precautions should be taken for patients with underlying conditions.

APDL1-CART cells exhibit strong PD-L1-specific activity against leukemia cells.

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.

Role and mechanism of PTEN in Burkitt's lymphoma.

The aim of the present study was to explore the possible mechanisms of phosphatase and tensin homolog (PTEN) in the pathogenesis of Burkitt's lymphoma, and provide novel information that can be used in the targeted treatment of this disease. PTEN lentiviral overexpression vector and short­hairpin PTEN silencing vectors were constructed. The effect of PTEN on the growth and proliferation of CA46 and RAJI cells was analyzed using a Cell Counting Kit­8 assay. Apoptosis was detected by Hoechst 33342 and propidium iodide double staining. Flow cytometry was used to analyze the cell cycle. A Transwell chamber was used to detect cell migration and invasion abilities. Western blot analysis was used to detect related protein changes. The mechanism of the effect of PTEN on the biological characteristics of Burkitt's lymphoma cells was subsequently analyzed. The results revealed that PTEN inhibited the proliferation of CA46 and RAJI cells by downregulating the expression of p­AKT, It was indicated that the upregulation of proapoptotic proteins (including Bad and Bax) induced apoptosis, regulated cyclin (including P53, P21, CDK4, CDK6, cyclin D3 and cyclin H) to inhibit cell cycle progression, and mediated epithelial­mesenchymal transition­like cell markers (including E­cadherin, N­cadherin, ß­catenin, TCF­8, vimentin, Slug and Snail) to inhibit cell migration and invasion. In conclusion, the tumor­suppressor gene PTEN inhibited the phosphoinositide 3­kinase/protein kinase B (PI3K/AKT) signaling pathway and inhibited the proliferation and migration of Burkitt's lymphoma cells, induced apoptosis and cell cycle arrest, thus playing a crucial role in the pathogenesis of Burkitt's lymphoma.

Calreticulin increases growth and progression of natural killer/T-cell lymphoma.

In this study, we investigated the role of calreticulin (CALR) in the pathogenesis of natural killer/T-cell lymphoma (NKTCL). CALR expression was significantly higher in the NKTCL tissues than normal control tissues in the GSE80632 dataset. High CALR expression correlated with poorer overall survival of NKTCL patients (P = 0.0248). CALR mRNA and protein levels were significantly higher in NKTCL cell lines (NK92, SNK6, and SNT8) than normal NK cells. CALR-silenced SNK6 cells generated significantly smaller xenograft tumors in immunodeficient NCG mice than control SNK6 cells. CALR-knockdown NKTCL cells showed significantly less in vitro proliferation and Transwell migration than the controls. CALR knockdown inhibited G1-to-S phase cell cycle progression by increasing the levels of p27 cell cycle inhibitor and reducing the levels of cyclin E2 and cyclin-dependent kinase 2 (CDK2). CALR knockdown inhibited epithelial-to-mesenchymal transition (EMT) by decreasing the levels of ß-catenin and TCF/ZEB1 and upregulating E-cadherin. These data demonstrate that CALR regulates the growth and progression of NKTCL cells by modulating G1-to-S cell cycle progression and EMT.

[Relationship between PANTR1 and Imatinib Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Related Mechanisms].

OBJECTIVE: To investigate the relationship between long non-coding RNA (LncRNA) PANTR1 and imatinib resistance in chronic myeloid leukemia cell line K562 and its mechanism. METHODS: K562 control cells (Control) and K562 imatinib resistant cells (ImR) were cultured. Two siRNA vectors targeting PANTR1 and control vectors were transfected into K562-ImR cells by lentivirus as ImR-siPA#1, ImR-siPA#2 and ImR-siControl cells, respectively. Imatinib semi-inhibitory concentration (IC50) was detected by CCK-8 kit. The expression level of PANTR1, BCR/ABL, MDR, CD44 and CD133 mRNA were detected by fluorescence quantitative PCR（RT-qPCR）, and the expression level of BCR/ABL, MDR, CD44 and CD133 protein were detected by Western blot. RESULTS: Imatinib IC50 in ImR cells was significantly higher than that in control cells (P＜0.01), that of ImR-siPA#1 and ImR-siPA#2 cells were significantly lower than that in ImR-sicontrol cells (P＜0.01), but still significantly higher than that in control cells (P＜0.01). The mRNA expression level of PANTR1, BCR/ABL, MDR, CD44 and CD133 in ImR cells were significantly higher than those in control cells (P＜0.01). The mRNA expression level of PANTR1, MDR, CD44 and CD133 in ImR-siPA#1 and ImR-siPA#2 cells were significantly lower than those in ImR-siControl cells (P＜0.01), while the expression level of BCR/ABL mRNA was not significantly different (P＞0.05). The protein expression level of BCR/ABL, MDR, CD44 and CD133 in ImR cells were significantly higher than those in control cells, while the protein expression level of MDR, CD44 and CD133 in ImR-siPA#1 and ImR-siPA#2 were significantly lower than those in ImR-siControl cells. CONCLUSION: LncRNA PANTR1 can promote the expression of MDR and stem cell marker in chronic myeloid leukemia cell line K562, and mediate imatinib resistance.

[Silencing Calreticulin Expression Inhibits Invasion Ability of SNK6 Cells in Vitro via Down-Regulating Expression of VEGF and MMP2/9].

OBJECTIVE: To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism. METHODS: The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot. RESULTS: The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced. CONCLUSION: The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.

Protective autophagy or autophagic death: effects of BEZ235 on chronic myelogenous leukemia.

PURPOSE: To investigate the effects of BEZ235 on chronic myeloid leukemia (CML) cells. METHODS: MTS assay was used to detect the proliferation of CML cells. The proteins expression were detected by Western blot assay. The effects of BEZ235 on autophagy in CML cells were verified through transmission electron microscopy and evaluated by laser confocal microscopy. Annexin V-FITC/PI double staining flow cytometry was used to detect apoptosis. A xenograft model was established to observe the therapeutic effect of BEZ235 in vivo. RESULTS: BEZ235 could inhibit the proliferation of CML cells; CQ and 3-MA could increase the proliferation inhibition and Z-VAD-FMK can reduce the proliferation inhibition of BEZ235 on CML cells (P<0.05). Results of TEM showed that the autophagosomes of CML cells treated with BEZ235 increased (P<0.05). The results by confocal microscopy showed that the autophagic activity of K562 cells increased with BEZ235 treatment. When BEZ235 combined with CQ, BEZ235-induced autophagic flow was blocked. FCM results showed that BEZ235 could induces apoptosis in CML cells. Z-VAD-FMK could decrease the apoptosis of CML cells induced by BEZ235. CQ increased the apoptosis of CML cells induced by BEZ235 (P<0.05). Western blot showed that BEZ235 inhibited the phosphorylation of AKT and S6K. BEZ235 alone could upregulate the expression of cleaved caspase-3 and LC3II. When combined with Z-VAD-FMK, the expression of cleaved caspase-3 was lower than that of BEZ235 alone. When combined with CQ, the expression of cleaved caspase-3 and LC3II were higher than those of BEZ235 alone (P<0.05). BEZ235 could inhibit the growth of xenografts of CML cell line. CONCLUSION: BEZ235 can inhibit the proliferation of CML cells, induce apoptosis, and enhance autophagy activity. It induces protective autophagy. The combination of CQ can enhance the apoptosis and proliferation inhibition of CML cells induced by BEZ235.

In vitro inducing effect of dendritic cells cotransfected with survivin and granulocyte-macrophage colony-stimulating factor on cytotoxic T cell to kill leukemic cells.

BACKGROUND: Survivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells. METHODS: After derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL. RESULTS: Expression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group. CONCLUSION: DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.

FLT3 inhibitors in acute myeloid leukemia.

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.

[Comparison of Laboratory Indexes and Therapeutic Efficacy between Two Kinds of Different RBC Transfusion in AIHA Patients].

OBJECTIVE: To investigate the effect of 2 kinds of red blood cells (RBC) on the laboratorial indexes and therapeutic efficacy of patients with autoimmune hemolytic anemia (AIHA). METHODS: The clinical data of 120 patients with AIHA from June 2015 to June 2016 were analyzed retrospectively. These 120 patients were divided into A goup and B group. The patients in A group (60 cases) were infused with washed RBC, while the patients in B group (60 cases) were infused with WBC-deplated RBC. The changes of laboratotial indexes, clinical symptoms and incidence of adverse reactions in 2 groups before treatment and at 24 hourse after treatment as well as the therapeutic efficacy and improvement status of clinical symptoms were compared. RESULTS: The RBC count and Hb level in 2 groups after treating for 1 day were significantly higher than those before treatment (P<0.01), while the TBIL level and reticulocyte (Ret) count were significantly lower than those before treatment (P<0.01). However, the RBC, Hb, TBIL levels and Ret count in 2 groups before and after treatment showed no statistical difference (P>0.05); the improvement of clinical symptoms, complex therapeutic efficacy and incidence of adverse reactions in 2 groups after treatment also showed no significantly difference (P>0.05). CONCLUSION: Both washed and leukocyte-deplated RBC can alleviate the anemia in patients with AIHA in short-time, but the leucocyte-deplated RBC is more expensive, suggesting that the wased RBC is more practical for treatment of AIHA patients.

[Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells].

OBJECTIVE: To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. RESULTS: Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. CONCLUSION: The rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G1/G0 phase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.