CD4+CD25+Foxp3+ regulatory T cells (Tregs) accumulate in bone marrow microenvironment in acute myeloid leukemia (AML). However, little is known about how the tumor environment including tumor cells themselves affects this process. Here we demonstrated that AML cells expressed inducible T-cell costimulator ligand (ICOSL) that can provide costimulation through ICOS for the conversion and expansion of Tregs sustaining high Foxp3 and CD25 expression as well as a suppressive function. TNF-a stimulation up-regulated the expression of ICOSL. Furthermore, both the conversion and expansion of CD4+CD25+Foxp3+ T cells and CD4+ICOS+Foxp3+ T cells were induced by co-culture with AML cells overexpressed ICOSL. CD4+CD25+ICOS+ T cells possessed stronger ability to secrete IL-10 than CD4+CD25+ICOS− T cells. The mechanism by which IL-10 promoted the proliferation of AML cells was dependent on the activation of the Akt, Erk1/2, p38, and Stat3 signaling pathways. Blockade of ICOS signaling using anti-ICOSL antibody impaired the generation of Tregs and retarded the progression of an AML mice model injected with C1498 cells. The expression of ICOSL of patient AML cells and ICOS+ Tregs were found to be predictors for overall survival and disease-free survival in patients with AML, with ICOS+ Treg cell subset being a stronger predictor than total Tregs. These results suggest that ICOSL expression by AML cells may directly drive Treg expansion as a mechanism of immune evasion and ICOS+ Treg cell frequency is a better prognostic predictor in patients with AML.

Pharmacological modulation of autophagy has been referred to as a promising therapeutic strategy for cancer. Matrine, a main alkaloid extracted from Sophora flavescens Ait, has antitumour activity against acute myelocytic leukaemia (AML). Whether autophagy is involved in antileukaemia activity of matrine remains unobvious. In this study, we demonstrated that matrine inhibited cell viability and colony formation via inducing apoptosis and autophagy in AML cell lines HL‐60, THP‐1 and C1498 as well as primary AML cells. Matrine promoted caspase‐3 and PARP cleavage dose‐dependently. Matrine up‐regulated the level of LC3‐II and down‐regulated the level of SQSTM1/p62 in a dose‐dependent way, indicating that autophagy should be implicated in anti‐AML effect of matrine. Furthermore, the autophagy inhibitor bafilomycin A1 relieved the cytotoxicity of matrine by blocking the autophagic flux, while the autophagy promoter rapamycin enhanced the cytotoxicity of matrine. Additionally, matrine inhibited the phosphorylation of Akt, mTOR and their downstream substrates p70S6K and 4EBP1, which led to the occurrence of autophagy. In vivo study demonstrated that autophagy was involved in antileukaemia effect of matrine in C57BL/6 mice bearing murine AML cell line C1498, and the survival curves showed that mice did benefit from treatment with matrine. Collectively, our findings indicate that matrine exerts antitumour effect through apoptosis and autophagy, and the latter one might be a potential therapeutic strategy for AML.

Intratumoral accumulation of CD4 + CD25 + Foxp3 + regulatory T (Treg) cells occurs in acute myeloid leukemia (AML), but little is known about the role of tumor cells themselves in this process. Here, we showed that an immune checkpoint PD-L1 expressed by AML cells promoted the conversion and expansion of Treg cells sustaining high expression of Foxp3 and PD-1 as well as a suppressive function. Furthermore, an AML cell line HEL overexpressed PD-L1 promoted the conversion and expansion of Treg cells and CD4 + PD-1 + Foxp3 + T (PD-1 + Treg) cells from the conventional CD4 + T cells. CD4 + CD25 high PD-1 + T cells secreted more IL-10 production than CD4 + CD25 high PD-1 − T cells. IL-35, another cytokine secreted by Treg cells, promoted the proliferation of HL-60 cells and enhanced chemoresistance to cytarabine. Blockade of PD-1 signaling using anti-PD-L1 antibody dramatically impaired the generation of Treg cells and sharply retarded the progression of a murine AML model injected with C1498 cells. The frequency of intratumoral PD-1 + Treg cells was capable of predicting patient survival in patients with AML. In conclusion, our data suggest that PD-L1 expression by AML cells may directly drive Treg cell expansion as a mechanism of immune evasion and the frequency of PD-1 + Treg cells is a potential prognostic predictor in patients with AML.

RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.

Introduction: B Cell Mature Antigen (BCMA)-targeted chimeric antigen receptor T (CAR-T) cell therapy emerges as promising treatment for patients with relapse/refractory multiple myeloma (RRMM). Previous studies indicate patients who receive high-dose CAR-T cells may achieve better remission but have worse adverse events, like cytokine release syndrome (CRS). To solve this dilemma, we have developed novel autologous CAR-T therapeutics CT053 that are genetically modified T cells comprising an extracellular anti-BCMA human scFv and an intracellular 4-1BB costimulatory motif connected to a CD3-zeta T cell activation domain. Methods: A multi-center investigator-initiated clinical study is designed to evaluate CT053 in patients with RRMM who have failed in the prior treatment with ≥2 regimens, including a proteasome inhibitor, an immunomodulatory agent, and anti-CD38 monoclonal antibody. All patients have ≥50% BCMA expression on malignant cells. Patients are subjected to the lymphodepletion with 20-25 mg/m2 fludarabine and 300-500 mg/m2 cyclophosphamide daily for 2-4 days prior to receiving single-dose infusion of CT053 CAR-T cells. In case of progressive disease, patient may be dosed again on basis of investigators' evaluation of the disease status, BCMA expression and CAR-T persistence. Most enrolled patients received a single dose of 1.5 x 108 cells, except for 1 patient who received 0.5 x 108 cells and 1 patient who was infused with 1.8 x 108 cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs) and CAR T related AEs. Additional outcome measures include clinical response assessed according to the IMWG Uniform Response Criteria for Multiple Myeloma, overall and progression-free survival, pharmacokinetics and pharmacodynamic of CT053. Results: The study was performed in compliance with the declaration of Helsinki. As of the data cut-off date (July 10th, 2018), 16 patients (median 55 [39 to 67] years old) with a median of 3.9 (0.4 to 16.7) years since MM diagnosis, were infused with CT053. Patients had a median of 4 prior different regimens (range 2 to 10), and 56% (9/16) patients received prior autologous or allogeneic stem cell transplant. Among 16 patients, no neurotoxicity and no dose-limiting toxicities (DLT) were observed. The most common grade≥3 CAR-T related AEs were 3 thrombocytopenia (19%), 3 leukopenia (19%), 2 anemia (13%), 2 neutropenia (13%), 2 fever (13%) (Figure 1A). CRS was reported in 3 patients, including 1 Grade 3, 1 Grade 2 and 1 Grade 1, who had rapid recovery after Tocilizumab administration. 13/16 patients were eligible for initial evaluation of early clinical response with a median observation period of 8 (4 to 36) weeks. Overall response rate (ORR) in 13/13 patients was 100% post treatment. 12/13 patients (92%) quickly achieved partial response (4 PR), very good PR (6 VGPR), and complete response (2 CR) within 4 weeks post single-dose infusion (Figure 1B). 5/12 patients (42%) who were dosed at ≥1.5 x 108 CT053 CAR-T cells obtained CR at a median of 8 weeks post treatment. Durable responses from 4 weeks towards the data cut-off date were found in 12/13 patients (92%). One relapse from VGPR by the Week 12 was reported in a patient who had aggressive RRMM at enrollment and received the reduced dose of lymphodepletion regimen at 19 mg/m2 fludarabine and 192 mg/m2 cyclophosphamide for 2 days prior to CT053 infusion. Because positive BCMA expression on malignant cells was verified at relapse, the patient was re-dosed with CT053 at the Week 16 and subjected to the further evaluation. All patients had detectable CAR-T expansion from Day 3 post CT053 infusion. Expansion peaks were found on Day 7 (5/13), Day 14 (6/13) and Day 21 (2/13). 11/13 patients had notable persistence of CT053 CAR-T cells up to 4-6 months. The only relapsed patient had the lowest CAR-T expansion peak among 13 patients, indicating the potential correlation between CAR-T expansion and response outcome. Conclusions: Data from this early-stage clinical study showed the unparalleled safety and efficacy of CT053 CAR-T cells. Major AEs were transient, manageable, and reversible. 100% ORR in 13/13 evaluable patients were reported post single-dose infusion of 0.5~1.8 x 108 cells. 5/12 patients who were dosed at ≥1.5 x 108 CAR-T cells rapidly achieved durable CR at median of 8 weeks, suggesting CT053 could be developed as competitive therapeutics to treat patients with RRMM. Disclosures Ruan: CARsgen Therapeutics: Employment. Xiao:CARsgen Therapeutics: Employment, Equity Ownership. Wang:CARsgen Therapeutics: Employment, Equity Ownership. Li:CARsgen Therapeutics: Employment, Equity Ownership.

It is generally believed that there is correlation between cancer prognosis and pretreatment PLR and NLR. However, there are limited data about their role in diffuse large B cell lymphoma (DLBCL). This study aims to determine the prognostic value of pretreatment PLR and NLR for patients who have DLBCL. The associations between clinical characteristics and NLR and PLR were evaluated among 182 DLBCL patients from January 2005 to June 2016. The optimal cutoff values for high PLR (⩾150) and NLR (⩾2.32) in prognosis prediction were determined. The effect of NLR and PLR on survival was evaluated through multivariate Cox regression analysis, univariate analysis, and log-rank test. According to the evaluation results, patients with high NLR and PLR had significantly shorter OS (P=0.026 and P=0.035) and PFS (P=0.024 and P=0.022) compared with those who have low PLR and NLR. On multivariate analyses, IPI>2, elevated LDH, and PLR⩾2.32 were prognostic factors for OS and PFS in DLBCL patients. Therefore, we demonstrated that high PLR and NLR predicted adverse prognostic factors in DLBCL patients.

This study examined the prognostic value of the baseline red blood cell distribution width (RDW) in diffuse large B cell lymphoma (DLBCL) patients. The associations between RDW and clinical characteristics were assessed in 161 DLBCL patients from 2005 to 2016. The log-rank test, univariate analysis, and Cox regression analysis were used to evaluate the relationship between RDW and survival. A RDW of 14.1% was considered to be the optimal cut-off value for predicting prognosis. A high RDW was associated with more frequent B symptoms (P=0.001), a higher International Prognostic Index score (P=0.032), more extranodal sites of disease (P=0.035), and significantly lower Eastern Cooperative Oncology Group performance status (P=0.031). The log-rank test demonstrated that patients with a high RDW had a shorter overall survival (OS) (2-year OS rate, 53.6% vs. 83.6%, P<0.001) and progression-free survival (PFS) (2-year PFS rate, 44.7% vs. 81.8%, P<0.001). The multivariate analysis demonstrated that RDW ≥14.1% was an independent predictor of OS (odds ratio [OR] = 0.345, P<0.001) and PFS (OR = 0.393, P=0.001). We demonstrated that a high RDW predicted an unfavorable prognosis in patients with DLBCL.

Accumulating evidence shows that tigecycline, a first‐in‐class glycylcycline, has potential antitumour properties. Here, we found that tigecycline dramatically inhibited the proliferation of multiple myeloma (MM) cell lines RPMI‐8226, NCI‐H929 and U266 in a dose and time‐dependent manner. Meanwhile, tigecycline also potently impaired the colony formation of these three cell lines. Mechanism analysis found that tigecycline led to cell cycle arrest at G0/G1 with down‐regulation of p21, CDK2 and cyclin D1, rather than induced apoptosis, in MM cells. Importantly, we found that tigecycline induced autophagy and an autophagy inhibitor bafilomycin A1 further amplified the tigecycline‐induced cytotoxicity, suggesting that autophagy plays a cytoprotective role in tigecycline‐treated MM cells. Mechanisms modulating autophagy found that tigecycline enhanced the phosphorylation of AMPK, but did not decrease the phosphorylation of Akt, to inhibit the phosphorylation of mTOR and its two downstream effectors p70S6K1 and 4E‐BP1. Tigecycline effectively inhibited tumour growth in the xenograft tumour model of RPMI‐8226 cells. Autophagy also occurred in tigecycline‐treated tumour xenograft, and autophagy inhibitor chloroquine and tigecycline had a synergistic effect against MM cells in vivo. Thus, our results suggest that tigecycline may be a promising candidate in the treatment of MM.