Outcomes in patients with multiple myeloma receiving salvage treatment after BCMA-specific CAR-T therapy: A retrospective analysis of LEGEND-2.

Chimeric antigen receptor T-cell (CAR-T) therapy targeting B-cell maturation antigen (BCMA) has shown profound efficacy and manageable toxicity in patients with relapsed/refractory multiple myeloma (RRMM). However, determining the best course of treatment for post-CAR-T therapy relapse remains a significant challenge. We conducted a retrospective analysis of patients from the phase I LEGEND-2 study (NCT03090659) enrolled at the Xi'an site, analysing the first salvage line of therapy and outcomes in patients with RRMM who progressed after receiving LCAR-B38M CAR-T therapy. Of 45 eligible patients, 34 (76%) had progressive disease (PD). Overall response rate (ORR) to salvage treatment was 50.0%. Median progression-free survival (PFS) after starting salvage treatment was 16.3 months. Median PFS of patients receiving proteasome inhibitor (PI)-based combination therapy was longer (28.2 months) than that of patients receiving a second BCMA CAR-T (including LCAR-B38M; 3.9 months, p = 0.0022) or chemotherapy (1.67 months, p = 0.0001). All patients with extramedullary disease at baseline (n = 11) progressed after CAR-T therapy; ORR to salvage therapy was 25.0% and median PFS was 9.7 months. In conclusion, salvage therapy in patients with PD after receiving LCAR-B38M CAR-T cells produced moderate efficacy, with better outcomes for PI-based salvage regimens.

A comprehensive analysis of coagulopathy during anti-B cell maturation antigen chimeric antigen receptor-T therapy in multiple myeloma, a retrospective study based on LEGEND-2.

Chimeric antigen receptor (CAR)-reprogrammed T cell therapy is a novel and powerful treatment against hematological malignancies. Cytokine release syndrome (CRS) and other potentially life-threatening toxicities are known side effects which need appropriate management and supportive care. Coagulopathy is a common and severe CAR-T-related adverse event, while a comprehensive profile of coagulopathy in patients with multiple myeloma (MM) undergoing CAR-T cell therapy has not been reported. Therefore, we performed a comprehensive analysis of coagulopathy in 51 patients with r/r MM given anti-B cell maturation antigen CAR-T cell therapy. We found that 49% of patients had coagulation disorders, and 29% of patients experienced disseminated intravascular coagulation (DIC). Severe CRS, abnormal liver function and higher tumor burden were risk factors for the CAR-T-related coagulopathy. We found that the serum IL-6 level and alanine aminotransferase level were potential indicators for CAR-T-related DIC. Furthermore, we found that coagulation disorders occurred within 1 month after CAR-T cell infusion, mainly between days 10 and 13, which was 2-5 days later than the beginning of CRS and simultaneous with the beginning of abnormal liver function and the peak of CRS. In addition, although patients with coagulation dysfunction had a trend for better outcomes and prognosis, no statistical significance was found. In conclusion, our research provided a comprehensive understanding of CAR-T-related coagulopathy in MM. Upon timely and standardized treatment, coagulopathy was manageable in most cases.

Expression profile analysis of long non-coding RNA in acute myeloid leukemia by microarray and bioinformatics.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt that are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in acute myeloid leukemia (AML), we analyzed the expression profile of lncRNAs and mRNAs in AML. Five pairs of AML patients and iron deficiency anemia (IDA) controls were screened by microarray. Through coexpression analysis, differently expressed transcripts were divided into modules, and lncRNAs were functionally annotated. We further analyzed the clinical significance of crucial lncRNAs from modules in public data. Finally, the expression of three lncRNAs, RP11-222K16.2, AC092580.4, and RP11-305O.6, were validated in newly diagnosed AML, AML relapse, and IDA patient groups by quantitative RT-PCR, which may be associated with AML patients' overall survival. Further analysis showed that RP11-222K16.2 might affect the differentiation of natural killer cells, and promote the immunized evasion of AML by regulating Eomesodermin expression. Analysis of this study revealed that dysregulated lncRNAs and mRNAs in AML vs IDA controls could affect the immune system and hematopoietic cell differentiation. The biological functions of those lncRNAs need to be further validated.

Circ-ANAPC7 is Upregulated in Acute Myeloid Leukemia and Appears to Target the MiR-181 Family.

BACKGROUND/AIMS: Circular RNAs (circRNAs) are a family of novel non-coding RNAs associated with various diseases, especially cancer. Recent studies have demonstrated that circRNAs participate in pathogenesis mainly by acting as microRNA (miRNA) sponges. The expression profile of circRNAs in acute myeloid leukemia (AML) has rarely been reported. METHODS: Profiles of circRNAs were analyzed using an Arraystar human circRNA microarray with 5 bone marrow samples from patients with newly diagnosed AML and 5 from patients with iron-deficiency anemia. Quantitative reverse transcription PCR was used to validate the expression pattern of circRNAs. Furthermore, circRNA-miRNA network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied. RESULTS: CircRNA microarray analysis revealed that 698 circRNAs were differentially expressed in AML patients, with 282 circRNAs found to be upregulated and 416 to be downregulated. Quantitative reverse transcription PCR showed that circ-ANAPC7 was significantly upregulated in AML. Bioinformatics analysis predicted that circ-ANAPC7 acts as a sponge for the miR-181 family, KEGG analysis revealed that it is associated with cancer-related pathways, and GO analysis indicated that most of its target genes are involved in biological processes. CONCLUSIONS: These findings show that circ-ANAPC7 is a promising biomarker for AML, and that it might participate in AML pathogenesis by acting as a sponge for the miR-181 family.

High-Intensity Chemotherapy is Associated with Better Prognosis in Young Patients with High-Risk Diffuse Large B-Cell Lymphoma: A 10-Year Single-Center Retrospective Cohort Study.

BACKGROUND Patients <60 years old with high-risk diffuse large B-cell lymphoma (DLBCL) receiving standard RCHOP(E) treatment display high relapse rates. Here, we compared this standard regimen to a high-intensity regimen in terms of recurrence and long-term survival. MATERIAL AND METHODS Newly diagnosed DLBCL patients <60 years old who were treated at the Second Hospital Affiliated with Xi'an Jiaotong University between January 2004 and December 2013 (n=198, 18-60 years) were included in the study. The high-intensity group included 107 patients (54.0%) who received >8 courses of chemotherapy (high-dose CHOP, CHOP-E, EPOCH, MAED, MMED, and HyperCVAD). The control group included 91 patients (46.0%) who received 6-8 courses of CHOP-based treatment. Response rate (RR), survival, relapse, and adverse effects were compared. RESULTS Baseline characteristics of the patients were similar between the 2 groups. Median follow-up was 64.5 months. RR in the high-intensity and control groups was 88.8% and 84.6% (P=0.387), respectively; 5-year overall survival was 66.4% and 36.3% (P<0.001), respectively; 5-year progression-free survival was 56.1% and 28.6% (P<0.001), respectively; 5-year disease-free survival was 54.2% and 24.2% (P<0.001), respectively; and relapse rate during follow-up was 29.5% and 67.5% (P<0.001), respectively. There were no significant differences in adverse effects between the 2 groups. CONCLUSIONS High-intensity chemotherapy is associated with better prognosis of patients <60 years old with newly diagnosed high-risk DLBCL.

Development and validation of a nomogram to predict poor efficacy of imatinib in the treatment of newly diagnosed chronic phase chronic myeloid leukemia patients.

Background: Imatinib is the most widely used tyrosine kinase inhibitor (TKI) in patients with newly diagnosed chronic-phase chronic myeloid leukemia(CML-CP). However, failure to achieve optimal response after imatinib administration, and subsequent switch to second-generation TKI therapy results in poor efficacy and induces drug resistance. In the present study, we developed and validated a nomogram to predict the efficacy of imatinib in the treatment of patients newly diagnosed with CML-CP in order to help clinicians truly select patients who need 2nd generation TKI during initial therapy and to supplement the risk score system. Methods: We retrospectively analyzed 156 patients newly diagnosed with CML-CP who met the inclusion criteria and were treated with imatinib at the Second Affiliated Hospital of Xi'an Jiao Tong University from January 2012 to June 2022. The patients were divided into a poor-response cohort (N = 60)and an optimal-response cohort (N = 43) based on whether they achieved major molecular remission (MMR) after 12 months of imatinib treatment. Using univariate and multivariate logistic regression analyses, we developed a chronic myeloid leukemia imatinib-poor treatment (CML-IMP) prognostic model using a nomogram considering characteristics like age, sex, HBG, splenic size, and ALP. The CML-IMP model was internally validated and compared with Sokal, Euro, EUTOS, and ELTS scores. Results: The area under the curve of the receiver operator characteristic curve (AUC)of 0.851 (95% CI 0.778-0.925) indicated satisfactory discriminatory ability of the nomogram. The calibration plot shows good consistency between the predicted and actual observations. The net reclassification index (NRI), continuous NRI value, and the integrated discrimination improvement (IDI) showed that the nomogram exhibited superior predictive performance compared to the Sokal, EUTOS, Euro, and ELTS scores (P < 0.05). In addition, the clinical decision curve analysis (DCA) showed that the nomogram was useful for clinical decision-making. In predicting treatment response, only Sokal and CML-IMP risk stratification can effectively predict the cumulative acquisition rates of CCyR, MMR, and DMR (P<0.05). Conclusion: We constructed a nomogram that can be effectively used to predict the efficacy of imatinib in patients with newly diagnosed CML-CP based on a single center, 10-year retrospective cohort study.

Long-term remission and survival in patients with relapsed or refractory multiple myeloma after treatment with LCAR-B38M CAR T cells: 5-year follow-up of the LEGEND-2 trial.

BACKGROUND: The autologous anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy LCAR-B38M has been approved for the treatment of relapsed and refractory multiple myeloma in many countries across the world under the name ciltacabtagene autoleucel. LEGEND-2 was the first-in-human trial of LCAR-B38M and yielded deep and durable therapeutic responses. Here, we reported the outcomes in LEGEND-2 after a minimal 5-year follow-up. METHODS: Participants received an average dose of 0.5 × 106 cells/kg LCAR-B38M in split or single unfractionated infusions after cyclophosphamide-based lymphodepletion therapy. Investigator-assessed response, survival, safety and pharmacokinetics were evaluated. RESULTS: Seventy-four participants enrolled and had a median follow-up of 65.4 months. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 21.0% and 49.1%, with progressive flattening of the survival curves over time. Patients with complete response (CR) had longer PFS and OS, with 5-year rates of 28.4% and 65.7%, respectively. Twelve patients (16.2%) remained relapse-free irrespective of baseline high-risk cytogenetic abnormality and all had normal humoral immunity reconstituted. An ongoing CR closely correlated with several prognostic baseline indices including favorable performance status, immunoglobulin G subtype, and absence of extramedullary disease, as well as a combination cyclophosphamide and fludarabine preconditioning strategy. Sixty-two (83.8%) suffered progressive disease (PD) and/or death; however, 61.1% of PD patients could well respond to subsequent therapies, among which, the proteasome inhibitor-based regimens benefited the most. Concerning the safety, hematologic and hepatic function recovery were not significantly different between non-PD and PD/Death groups. A low rate of second primary malignancy (5.4%) and no severe virus infection were observed. The patients who tested positive for COVID-19 merely presented self-limiting symptoms. In addition, a sustainable CAR T population of one case with persistent remission was delineated, which was enriched with indolently proliferative and lowly cytotoxic CD4/CD8 double-negative functional T lymphocytes. CONCLUSIONS: These data, representing the longest follow-up of BCMA-redirected CAR T-cell therapy to date, demonstrate long-term remission and survival with LCAR-B38M for advanced myeloma. TRIAL REGISTRATION: LEGEND-2 was registered under the trial numbers NCT03090659, ChiCTRONH-17012285.

Serum soluble BCMA can be used to monitor relapse of multiple myeloma patients after chimeric antigen receptor T-cell immunotherapy.

PURPOSE: Chimeric antigen receptor T-cell (CAR-T) therapy has been proven very effective in treating hematologic malignancies. Ciltacabtagene autoleucel (cilta-cel), a second-generation CAR-T cell with double B cell maturation antigen (BCMA) targeting binding domains, showed an 88% overall response rate (ORR) in patients with relapsed/refractory multiple myeloma (MM), which were carried out in our institute. This study aimed to assess the prognostic potential of soluble BCMA (sBCMA) in serum as a biomarker in MM after CAR-T therapy. PATIENTS AND METHODS: Serum samples (n = 44) from MM patients were collected before and after CAR-T therapy. The level of sBCMA was analyzed by enzyme-linked immunosorbent assay (ELISA). Additionally, three patients' long-term longitudinal analysis were performed. RESULTS: Serum sBCMA level was correlated with the percentage of malignant plasma cells in bone marrow (r = 0.613). After CAR-T infusion, the sBCMA level in serum of MM patients decreased markedly (median: 508,513 pg/mL before CAR-T infusion, 89,198 pg/mL in the first month, 8448 pg/mL in the second months, and 6010 pg/mL in the third month after CAR-T infusion). In patients who obtained objective response (≥ PR), re-elevated sBCMA indicated the possibility of disease recurrence. At a cutoff 69,326.27 pg/mL, sBCMA shows high sensitivity (87.5%) and specificity (88.5%) for identifying relapse of MM after CAR-T therapy. CONCLUSION: Our results suggested that serum sBCMA level changes in response to the clinical status of MM patients after anti-BCMA CAR-T therapy. Furthermore, sBCMA may be a auxiliary biomarker for disease monitoring in MM patients after CAR-T therapy.

Phase II, Open-Label Study of Ciltacabtagene Autoleucel, an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor-T-Cell Therapy, in Chinese Patients With Relapsed/Refractory Multiple Myeloma (CARTIFAN-1).

PURPOSE: CARTIFAN-1 aimed to evaluate the efficacy and safety of ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen-targeting chimeric antigen receptor T-cell therapy, in Chinese patients with relapsed/refractory multiple myeloma (RRMM). METHODS: This pivotal phase II, open-label study (ClinicalTrials.gov identifier: NCT03758417), conducted across eight sites in China, enrolled adult patients with RRMM who had received ≥ 3 lines of prior therapy, including a proteasome inhibitor and immunomodulatory drug. Patients received a single infusion of cilta-cel (target dose 0.75 × 106 chimeric antigen receptor-positive viable T cells/kg). The primary end point was overall response rate. Secondary end points included progression-free survival (PFS), overall survival (OS), and incidence and severity of adverse events (AEs). RESULTS: As of the clinical cutoff of July 19, 2021, 48 patients received a cilta-cel infusion. At an 18-month median follow-up, the overall response rate was 89.6% (95% CI, 77.3 to 96.5), with a median time to first response of approximately 1 month; 77.1% of patients (95% CI, 62.7 to 88.0) achieved complete response or better. Medians for duration of response, PFS, and OS were not reached. The 18-month PFS and OS rates were 66.8% (95% CI, 49.4 to 79.4) and 78.7% (95% CI, 64.0 to 88.0), respectively. Hematologic AEs were common, including anemia (100%), neutropenia (97.9%), lymphopenia (95.8%), and thrombocytopenia (87.5%). Cytokine release syndrome occurred in 97.9% of patients (35.4% grade 3/4); the median time to onset was 7 days, and the median duration was 5 days. Infections occurred in 85.4% of patients (37.5% grade 3/4). Ten deaths occurred after cilta-cel infusion, eight of which were due to treatment-related AEs. CONCLUSION: These data demonstrate a favorable risk-benefit profile for a single infusion of cilta-cel, resulting in early, deep, and durable responses in heavily pretreated patients with RRMM in China.

Multidisciplinary recommendations for the management of CAR-T recipients in the post-COVID-19 pandemic era.

The outbreak of coronavirus disease 2019 (COVID-19) posed an unprecedented challenge on public health systems. Despite the measures put in place to contain it, COVID-19 is likely to continue experiencing sporadic outbreaks for some time, and individuals will remain susceptible to recurrent infections. Chimeric antigen receptor (CAR)-T recipients are characterized by durable B-cell aplasia, hypogammaglobulinemia and loss of T-cell diversity, which lead to an increased proportion of severe/critical cases and a high mortality rate after COVID-19 infection. Thus, treatment decisions have become much more complex and require greater caution when considering CAR T-cell immunotherapy. Hence, we reviewed the current understanding of COVID-19 and reported clinical experience in the management of COVID-19 and CAR-T therapy. After a panel discussion, we proposed a rational procedure pertaining to CAR-T recipients with the aim of maximizing the benefit of CAR-T therapy in the post COVID-19 pandemic era.

Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes.

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore, B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together, these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression, which may be associated with disease progression in MDS.

Neuronal Nitric Oxide Synthase Regulates Depression-like Behaviors in Shortening-Induced Obese Mice.

Shortening is mainly derived from the partial hydrogenation of palm oil and widely used in fast food. Food processed with shortening contains high levels of industrial trans fatty acids. Studies have shown that there is a correlation between industrial trans fatty acids, obesity, and depression. However, the regulatory effect of neuronal nitric oxide synthase (nNOS) on depression in obese patients is still unknown. The purpose of this study was to explore mood changes in obese mice fed a high shortening diet, and to determine the regulatory effect of nNOS on depressive-like behaviors in obese mice. We used a high shortening diet-induced obesity mouse model to systematically assess the metabolic response, behavioral changes, prefrontal and hippocampal nNOS protein levels, and the effect of nNOS inhibitors (7-nitroindole) on depression-like behavior in obese mice. Interestingly, obese mice on a 9-week high-shortening diet developed short-term spatial working memory impairment and anxiety-like behavior, and obesity may be a risk factor for cognitive impairment and mood disorders. In animals fed a high shortening diet for 12 weeks, obese mice developed depression-like behavior and had significantly elevated levels of nNOS protein expression in the hippocampus and prefrontal lobe. Administration of the nNOS inhibitor 7-nitroindole could improve depression-like behaviors in obese mice, further suggesting that inhibition of nNOS is helpful for depression associated with obesity.

Five-year remission without disease progression in a patient with relapsed/refractory multiple myeloma with extramedullary disease treated with LCAR-B38M chimeric antigen receptor T cells in the LEGEND-2 study: a case report.

BACKGROUND: Multiple myeloma remains incurable despite treatment advancements over the last 20 years. LCAR-B38M Cells in Treating Relapsed/Refractory Multiple Myeloma was a phase 1, first-in-human, investigator-initiated study in relapsed/refractory multiple myeloma conducted at four sites in China. The study used LCAR-B38M chimeric antigen receptor-T cells expressing two B-cell maturation antigen-targeting single-domain antibodies designed to confer avidity, and a CD3ζ signaling domain with a 4-1BB costimulatory domain to optimize T-cell activation and proliferation. This chimeric antigen receptor construct is identical to ciltacabtagene autoleucel. In the LEGEND-2 study (n = 57, Xi'an site), overall response rate was 88%; median (95% CI) progression-free survival and overall survival were 19.9 (9.6-31.0) and 36.1 (26.4-not evaluable) months, respectively; and median follow-up was 25 months. This case study reports on a patient with relapsed/refractory multiple myeloma (λ light chain type) who was treated with LCAR-B38M chimeric antigen receptor T cells in the LEGEND-2 study (Xi'an site); he had received five prior lines of treatment and had extensive extramedullary lesions. CASE PRESENTATION: The patient, a 56-year-old Asian male, received cyclophosphamide (500 mg daily × 3 days) as lymphodepletion therapy and a total dose of 0.5 × 106 chimeric antigen receptor + T cells/kg split into three infusions (days 1, 24, and 84 from June to August 2016). He experienced grade 2 cytokine release syndrome after the first infusion; all symptoms resolved with treatment. No cytokine release syndrome occurred following the second and third infusions. His λ light chain levels decreased and normalized 20 days after the first infusion, and extramedullary lesions were healed as of January 2018. He has sustained remission for 5 years and received no other multiple myeloma treatments after LCAR-B38M chimeric antigen receptor T cell infusion. As of 30 October 2020, the patient is still progression-free and has maintained minimal residual disease-negative (10-4) complete response status for 52 months. CONCLUSIONS: This case provides support that treatment with LCAR-B38M chimeric antigen receptor T cells can result in long-term disease remission of 5 or more years without disease progression in a heavily pretreated patient with extensive extramedullary disease and no other treatment options.

Four-year follow-up of LCAR-B38M in relapsed or refractory multiple myeloma: a phase 1, single-arm, open-label, multicenter study in China (LEGEND-2).

BACKGROUND: LCAR-B38M is a chimeric antigen receptor T cell product with two binding domains targeting B cell maturation antigen. Our previous reports showed a remarkable efficacy of LCAR-B38M in patients with relapsed/refractory multiple myeloma (RRMM) at a median follow-up of 2 years. Here, we report long-term safety and efficacy data from a median follow-up of 4 years. METHODS: LEGEND-2 was a phase 1, single-arm, open-label study conducted in four registered sites in China. Seventy-four participants with RRMM received LCAR-B38M treatment. Lymphodepletion was performed using cyclophosphamide or cyclophosphamide plus fludarabine. LCAR-B38M, at a median dose of 0.513 × 106 cells/kg, was intravenously administered either in three split infusions or in a single infusion. The primary objective was the safety of LCAR-B38M, and the secondary objective was efficacy. RESULTS: As of May 25, 2021, the median follow-up was 47.8 months. All patients experienced ≥ 1 adverse events (AEs). Grade ≥ 3 AEs were observed in 45/74 (60.8%) patients. Cytokine release syndrome (CRS) occurred in 68/74 (91.9%) cases; 7 (9.5%) had grade ≥ 3 CRS. One patient experienced grade 1 central nervous system toxicity. The overall response rate was 87.8%. Fifty-four out of 74 (73.0%) patients achieved complete response. The median progression-free survival was 18.0 months, and the median overall survival for all patients was not reached. The median duration of response was 23.3 months. Four patients experienced viral infection more than 6 months post-infusion, and four patients developed second primary non-hematological malignancies at a median time of 11.5 months post-CAR-T cell transfer. CONCLUSIONS: The 4-year follow-up data of LCAR-B38M therapy demonstrated a favorable long-term safety profile and a durable response in patients with RRMM. Trial registration Clinicaltrials.gov NCT03090659 (retrospectively registered on March 27, 2017); ChiCTR-ONH-17012285.

Novel neuroprotectant chiral 3-n-butylphthalide inhibits tandem-pore-domain potassium channel TREK-1.

AIM: To study the effects of 3-n-butylphthalide (NBP) on the TREK-1 channel expressed in Chinese hamster ovary (CHO) cells. METHODS: Whole-cell patch-clamp recording was used to record TREK-1 channel currents. The effects of varying doses of l-NBP on TREK-1 currents were also observed. Current-clamp recordings were performed to measure the resting membrane potential in TREK-1-transfected CHO (TREK-1/CHO) and wild-type CHO (Wt/CHO) cells. RESULTS: l-NBP (0.01-10 µmol/L) showed concentration-dependent inhibition on TREK-1 currents (IC(50)=0.06±0.03 µmol/L), with a maximum current reduction of 70% at a concentration of 10 µmol/L. l-NBP showed a more potent inhibition on TREK-1 current than d-NBP or dl-NBP. This effect was partially reversed upon washout and was not voltage-dependent. l-NBP 10 µmol/L elevated the membrane potential in TREK-1/CHO cells from -55.3 mV to -42.9 mV. However, it had no effect on the membrane potential of Wt/CHO cells. CONCLUSION: 1-NBP potently inhibited TREK-1 current and elevated the membrane potential, which may contribute to its neuroprotective activity.

lncRNA ST3GAL6­AS1 promotes invasion by inhibiting hnRNPA2B1­mediated ST3GAL6 expression in multiple myeloma.

Multiple myeloma (MM) is an incurable disease caused by the infiltration of malignant plasma B cells into bone marrow, whose pathogenesis remains largely unknown. Long non­coding RNAs (lncRNAs) have emerged as important factors in pathogenesis. Our previous study validated that lncRNA ST3 ß­galactoside α­2,3­sialyltransferase 6 antisense RNA 1 (ST3GAL6­AS1) was upregulated markedly in MM. Therefore, the aim of the study was to investigate the molecular mechanisms of ST3GAL6­AS1 in MM cells. ST3GAL6­AS1 expression levels in MM cells was detected using reverse transcription­quantitative PCR. ST3GAL6­AS1 antisense oligonucleotides and small interfering RNAs were transfected into MM cells to downregulate expression. In vitro assays were performed to investigate the functional role of ST3GAL6­AS1 in MM cells. RNA pull­down, RNA immunoprecipitation and comprehensive identification of RNA­binding proteins using mass spectrometry assays were used to determine the mechanism of ST3GAL6­AS1­mediated regulation of underlying targets. It was reported that knockdown of ST3GAL6­AS1 suppressed the adhesion, migration and invasion ability of MM cells in vitro. Expression of ST3GAL6 was significantly reduced when ST3GAL6­AS1 was knock downed in MM cells. Moreover, mechanistic investigation showed that ST3GAL6­AS1 could suppress ST3GAL6 mRNA degradation via interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). The present results suggested that upregulated lncRNA ST3GAL6­AS1 promotes adhesion and invasion of MM cells by binding with hnRNPA2B1 to regulate ST3GAL6 expression.

Effects of CpG oligodeoxynucleotides on the differentiation of Treg/Th17 cells.

AIM: The balance between Th17 cells and T regulatory (Treg) cells has emerged as a prominent factor in regulating cancer development. However, the effect of CpG oligodeoxynucleotides (ODNs) on the differentiation of Treg/Th17 cells has not been well studied. We sought here to explore the function of CpG ODNs in the differentiation of Tregs and Th17 cells in vitro and in vivo. METHODS: Mouse spleen cells were cultured with anti-CD3 monoclonal antibodies in vitro. Tregs and Th17 cell differentiation was induced by transforming growth factor (TGF)-ß and interleukin (IL)-2, or TGF-ß, IL-6, and IL-23, respectively. Then cells were treated with two CpG ODNs, CpG 1982, or CpG 1826. FBL-3-inoculated C57Bl/6 mice were treated with CpG 1826, tumor vaccine, or combination of CpG 1826 and tumor vaccine. After treatment, spleen cells and serum were isolated, and Tregs/Th17 cells were detected by flow cytometry. The expression of forkhead box P3 (Foxp3), retinoid-related orphan receptor gamma-t (RORγt), IL-10, and IL-17 mRNA was measured by real-time PCR, and protein levels were measured by Western blot and enzyme-linked immunosorbent assay. RESULTS: The frequency of Treg cells increased significantly (p < 0.05) in the FBL-3-inoculated leukemia mouse model compared with control mice, whereas the frequency of Th17 cells did not change. Median survival of mice after treatment with CpG 1826 and tumor vaccine was significantly prolonged compared with that of control mice (p < 0.05). The frequency of induced Treg cells decreased after treatment with CpG 1826, whereas the frequency of Th17 cells induced by cytokines in vitro and in the murine leukemia model increased following treatment with CpG 1826. Furthermore, after treatment with CpG 1826, the mRNA and protein levels of Foxp3 and IL-10 decreased significantly both in vitro and in vivo (p < 0.05), whereas those of RORγt and IL-17 increased significantly (p < 0.05). CONCLUSION: CpG 1826 may inhibit the differentiation of Treg cells induced by cytokines, promote the differentiation of Th17 cells in vitro and in murine leukemia models, and prolong the median survival of mice with leukemia.

MLAA-34 knockdown shows enhanced antitumor activity via JAK2/STAT3 signaling pathway in acute monocytic leukemia.

MLAA-34 is a novel leukemia-associated gene closely related to the carcinogenesis of acute monocytic leukemia (AML). MLAA-34 over expression has been observed to inhibit apoptosis in vitro. JAK2/STAT3 pathway plays an important role in cell proliferation, differentiation and inhibition of apoptosis in number of cancers. However, the relationship and interaction between MLAA-34 and JAK2/STAT3 has never been investigated in AML. This study investigates and reports a novel relationship between MLAA-34 and JAK2/STAT3 pathway in AML both in vitro and in vivo. We constructed MLAA-34 knockdown vector and transfected U937 cells to observe its apoptotic activities in relation to JAK2/STAT3 signaling pathway in vitro and then in vivo in mouse model. Levels of expression of MLAA-34 and JAK2/STAT3 and its downstream targets were also measured in AML patients and a few volunteers. We found that MLAA-34 knockdown increased U937 apoptosis in vitro and inhibited tumor growth in vivo. Components of the canonical JAK2/STAT3 pathway or its downstream targets, including c-myc, bcl-2, Bax, and caspase-3, were shown to be involved in the carcinogenesis of AML. We also found that the JAK2/STAT3 pathway positively regulated MLAA-34 expression. We additionally identified a STAT3 binding site in the MLAA-34 promoter where STAT3 binds directly and activates MLAA-34 expression. In addition, MLAA-34 was found to form a complex with JAK2 and was enhanced by JAK2 activation. Correlation of MLAA-34 and JAK2/STAT3 was further confirmed in AML patients. In conclusion, MLAA-34 is a novel regulator for JAK2/STAT3 signaling, and in turn, is regulated by this interaction in a positive feedback loop. Thus we report a novel model of interaction mechanism between MLAA-34 and JAK2/STAT3 which can be utilized as a potential target for a novel therapeutic approach in AML.

Epigallocatechin gallate inhibits cell growth and regulates miRNA expression in cervical carcinoma cell lines infected with different high-risk human papillomavirus subtypes.

The aim of the present study was to investigate the inhibitory effects of the polyphenol epigallocatechin-3-gallate (EGCG) on the growth of cervical carcinoma cell lines infected with different high-risk human papillomavirus (HPV) subtypes, as well as the associated regulation of microRNA (miR) expression. Cell proliferation was measured using an MTT assay. The effects of 7 different concentrations of EGCG (100, 80, 60, 40, 20, 10 and 0 µg/ml) on HeLa cell proliferation were assessed. HeLa cell growth was significantly inhibited by EGCG in a dose- and time-dependent manner (P<0.05), and the IC50 was 90.74 and 72.74 µg/ml at 24 and 48 h, respectively. The expression of miR-210, miR-29a, miR-203 and miR-125b in HeLa (HPV16/18+), SiHa (HPV16+), CaSki (HPV16+) and C33A (HPV-) cell lines was measured using quantitative polymerase chain reaction analysis. In CA33 cells, miR-203 (all P<0.001) and miR-125b (P<0.01 and <0.0001) were significantly downregulated by EGCG, and miR-210 was significantly upregulated with 40 and 60 µg/ml EGCG (P<0.0001). miR-125b was significantly downregulated (P<0.001 and <0.0001), and miR-210 and miR-29 were significantly upregulated by ≤80 µg/ml EGCG in HeLa cells (all P<0.0001). In CaSki cells, miR-210, miR-29a (all P<0.001) and miR-125b (P<0.01-0.0001) were significantly upregulated by EGCG. In SiHa cells, miR-125b (both P<0.001) and miR-203 (P<0.01 and <0.0001) were significantly upregulated by EGCG. In conclusion, the results of the present study suggest that EGCG suppresses cervical carcinoma cell growth, possibly via regulating the expression of miRs, suggesting their potential as therapeutic targets for the control and prevention of cervical cancer. Additionally, EGCG may be considered a novel anti-cervical cancer drug in the future.

[Cloning of New Antigen Gene MLAA-34 Promoter and Identification of Core Region in Acute Monocytic Leukemia].

OBJECTIVE: To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. METHODS: The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. RESULTS: The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P＜0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. CONCLUSION: The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.