Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

GPCR signaling contributes to immune characteristics of microenvironment and process of EBV-induced lymphomagenesis.

Epstein-Barr virus (EBV) is the oncogenic driver of multiple cancers. However, the underlying mechanism of virus-cancer immunological interaction during disease pathogenesis remains largely elusive. Here we reported the first comprehensive proteogenomic characterization of natural killer/T-cell lymphoma (NKTCL), a representative disease model to study EBV-induced lymphomagenesis, incorporating genomic, transcriptomic, and in-depth proteomic data. Our multi-omics analysis of NKTCL revealed that EBV gene pattern correlated with immune-related oncogenic signaling. Single-cell transcriptome further delineated the tumor microenvironment as immune-inflamed, -deficient, and -desert phenotypes, in association with different setpoints of cancer-immunity cycle. EBV interacted with transcriptional factors to provoke GPCR interactome (GPCRome) reprogramming. Enhanced expression of chemokine receptor-1 (CCR1) on malignant and immunosuppressive cells modulated virus-cancer interaction on microenvironment. Therapeutic targeting CCR1 showed promising efficacy with EBV eradication, T-cell activation, and lymphoma cell killing in NKTCL organoid. Collectively, our study identified a previously unknown GPCR-mediated malignant progression and translated sensors of viral molecules into EBV-specific anti-cancer therapeutics.

Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.

Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces. However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.

A nuclear NKRF interacting long noncoding RNA controls EBV eradication and suppresses tumor progression in natural killer/T-cell lymphoma.

Long intergenic noncoding RNAs (lincRNAs) are differentially expressed in EBV-infected cells and play an essential role in tumor progression. Molecular pathogenesis of lincRNAs in EBV-driven natural killer T cell lymphoma (NKTCL) remains unclear. Here we investigated the ncRNA profile using high-throughput RNA sequencing data of 439 lymphoma samples and screened out LINC00486, whose downregulation was further validated by quantitative real-time polymerase chain reaction in EBV-encoded RNA (EBER)-positive lymphoma, particularly NKTCL. Both in vitro and in vivo studies revealed the tumor suppressive function of LINC00486 through inhibiting tumor cell growth and inducing G0/G1 cell cycle arrest. As mechanism of action, LINC00486 specifically interacted with NKRF to abrogate its binding with phosphorylated p65, activated NF-κB/TNF-α signaling and subsequently enhanced EBV eradication. Solute carrier family 1 member 1 (SLC1A1), upregulated and mediated the glutamine-addiction and tumor progression in NKTCL, was negatively correlated with the expression of NKRF. NKRF specifically bound to the promoter and transcriptionally downregulated the expression of SLC1A1, as evidenced by Chromatin Immunoprecipitation (ChIP) and luciferase assay. Collectively, LINC00486 functioned as a tumor suppressor and counteracted EBV infection in NKTCL. Our study improved the knowledge of EBV-driven oncogenesis in NKTCL and provided the clinical rationale of EBV eradication in anti-cancer treatment.

[Application regularity and solution strategies of mineral medicine Cinnabaris in pediatric preparations].

Mineral medicine is a characteristic element of advantage of traditional Chinese medicine(TCM), which embodies unique scientific connotation. Cinnabaris is a characteristic drug in Chinese medicinal preparations, especially in Chinese medicinal pediatric preparations. Because of the adverse reactions caused by mercury contained, the safety and application of Cinnabaris have attracted much attention. To explore the application regularity and the value of the pediatric preparations containing Cinnabaris, this study statistically analyzed 32 Cinnabaris-contained pediatric preparations in the 2020 edition of the Chinese Pharmacopoeia and 105 pediatric preparations containing Cinnabaris in the Dictionary of Traditional Chinese Medicine Prescriptions(Vol. Ⅰ and Ⅱ). The statistical results indicated that the pediatric preparations and formulae containing Cinnabaris had great advantages in the treatment of pediatric convulsions, but there were still problems in dosage form, dosage, and quality control. In this study, ICP-MS and LC-AFS were further used to determine the content of total mercury and soluble mercury in 15 commercially available pediatric preparations containing Cinnabaris. It was found that the total mercury content was far higher than soluble mercury content in the sample preparations, and there was no obvious correlation between them. According to the results, the research and application strategies of Cinnabaris were put forward in order to provide references for the rational application of Cinnabaris in pediatric preparations.

[Expression of miR-106b-5p in children with primary immune thrombocytopenia and its correlation with T cells]. / å„¿ç«¥åŽŸå‘å ç–«æ€§è¡€å°æ¿å‡å°‘ç—‡miR-106b-5pçš„è¡¨è¾¾åŠå ¶ä¸ŽTç»†èƒžçš„ç›¸å ³æ€§ç ”ç©¶.

OBJECTIVES: To study the expression level of plasma miR-106b-5p in primary immune thrombocytopenia (ITP) and its correlation with the levels of T helper 17 cell (Th17) and regulatory T cell (Treg) and the Th17/Treg ratio. METHODS: A total of 79 children with ITP (ITP group) and 40 healthy children (control group) were selected as subjects. According to the treatment response, the 79 children with ITP were divided into three groups: complete response (n=40), partial response (n=18), and non-response (n=21). Quantitative real-time PCR was used to measure the expression level of miR-106b-5p. Flow cytometry was used to measure the frequencies of Th17 and Treg, and the Th17/Treg ratio was calculated. The correlation of the expression level of plasma miR-106b-5p with the frequencies of Th17 and Treg and the Th17/Treg ratio was analyzed. RESULTS: Compared with the control group, the ITP group had significantly higher levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significantly lower level of Treg (P<0.05). After treatment, the ITP group had significant reductions in the levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significant increase in the level of Treg (P<0.05). Compared with the partial response and non-response groups, the complete response group had significantly lower levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significantly higher level of Treg (P<0.05). The correlation analysis showed that in the children with ITP, the expression level of plasma miR-106b-5p was positively correlated with the Th17 level and the Th17/Treg ratio (r=0.730 and 0.816 respectively; P<0.001) and was negatively correlated with the Treg level (r=-0.774, P<0.001). CONCLUSIONS: A higher expression level of miR-106b-5p and Th17/Treg imbalance may be observed in children with ITP. The measurement of miR-106b-5p, Th17, Treg, and Th17/Treg ratio during treatment may be useful to the evaluation of treatment outcome in children with ITP.

Biosynthesis of a novel ganoderic acid by expressing CYP genes from Ganoderma lucidum in Saccharomyces cerevisiae.

Ganoderic acids (GAs), a group of highly oxygenated lanostane-type triterpenoids from the traditional Chinese medicinal mushroom Ganoderma lucidum, possessed significant pharmacological activities. Due to the difficulty in its genetic manipulation, low yield, and slow growth of G. lucidum, biosynthesis of GAs in a heterologous host is a promising alternative for their efficient production. Heterologous production of a GA, 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA), was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA to biosynthesize other GAs remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new GA 3,28-dihydroxy-lanosta-8,24-dien-26-oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by UPLC-APCI-HRMS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmids pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR, and hygromycin-resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418-resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work enriches the GA biosynthetic enzyme library, and is helpful to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum. KEY POINTS: • Another P450 gene responsible for GA's post-modification was discovered and identified. • One new GA, DHLDOA, was identified and produced via engineered yeast. • With the balance of the two CYP genes expression, DHLDOA production was significantly improved.

An Analysis of the Correlation Between Clinical Indexes and Pathological Classifications in 202 Patients with Lupus Nephritis.

OBJECTIVE: To investigate the correlation between clinical indexes and pathological classifications in 202 patients with lupus nephritis (LN). METHODS: A total of 202 LN cases were retrospectively analyzed. All these patients met the four diagnostic criteria for systemic lupus erythematosus (SLE) of the American College of Rheumatology revised in 1997. The pathological diagnostic criteria of LN were in accordance with the pathological LN classification revised by the International Society of Nephrology and the Society of Kidney Pathology in 2003. The patients were scored according to the improved SLE Disease Activity Index 2000 (SLEDAI-2K), and their basic data, clinical data, laboratory data, and pathological data were collected. RESULTS: Among the 202 patients, the ratio of male to female was 1:5.73, and type IV was the most common pathological LN classification. There were differences in the urine analysis, hypertension incidence, blood cell analysis, blood lipids, renal function, plasma albumin, immunological indexes, renal pathological score among the different pathological types (P < 0.05). In the early finding of renal function damage of the patients, cystatin C sensitivity was significantly higher than that of serum creatinine and blood urea nitrogen. Multiple linear regression analysis show that there are strong correlations between AI and SLEDAI, 24hU-Pr, serum C3, serum ALB, BUN, creatinine, UA and PLT (P < 0.001); and there are correlations between AI and serum IgM, IgA, C4, TC and LDL-C (P < 0.05). CONCLUSION: There is a clear correlation between pathological classifications and clinical indexes of LN. TRIAL REGISTRATION: Shen-PJ-2018-40, Study on Clinical and Molecular Mechanism of SLE.

[Effect of MiR-96 on Cell Invasion and Apoptosis in Pediatric Acute Myeloid Leukemia via Regulating MYB].

OBJECTIVE: To analyze the relationship of the expression of transcription factor MYB targeted regulation by miR-96 to cell invasion and apoptosis in pediatric acute myeloid leukemia (AML). METHODS: A total of 65 children with AML in The 928 Hospital of PLA Joint Logistics Support Forces from January 2017 to November 2019 were selected, including 35 cases diagnosed as primary AML and 30 cases as complete remission AML. Thirty children with immune thrombocytopenia were selected as control group. The clinical characteristics were analyzed and compared between the two groups. The levels of miR-96 and MYB in peripheral blood samples were detected by qRT-PCR and compared between the two groups. The miR-96 mimics and its negative control (NC), inhibitor-miR-96 and its NC transfected HL60 cells induced by liposome (Lipofectamine 2000), respectively, Then the expression levels of MYB were detected with Western blot and compared among four HL60 cell groups. The invasion ability of four HL60 cell groups were detected with Transwell assay. The cell proliferation ability of four HL60 cell groups were detected with MTT at 24 h, 48 h, and 72 h, respectively. The apoptosis rates of four HL60 cell groups were detected with flow cytometry. RESULTS: Compared with control group, the level of miR-96 in AML children were higher, but MYB lower (P<0.05). Compared with complete remission AML, the level of miR-96 in primary AML was higher, but MYB lower (P<0.05). Western blot analysis showed that, the expression level of MYB in the four HL60 cell groups was different (P<0.05), the lowest was in miR-96 mimics group, followed by miR-96 NC group and inhibitor-miR-96 NC group, and the highest in inhibitor-miR-96 group (P<0.05), while there was no difference between miR-96 NC group and inhibitor-miR-96 NC group (P>0.05). The promotion of over-expression level of miR-96 on the invasion ability of HL 60 cells was confirmed by Transwell assay. MTT assay showed that miR-96 could promote the proliferation of HL60 cells, inhibit the apoptosis of HL60 cells, and the effect was time-dependent manner (r=0.804). The inhibition of miR-96 on HL60 cells apoptosis was also confirmed with flow cytometry. CONCLUSION: MiR-96 has significant negative effect on invasion and apoptosis of AML cells by targeting regulation MYB, and it might be a potential novel strategy for pediatric AML treatment.

Biosynthesis of a ganoderic acid in Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganoderma lucidum.

Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of cytochrome P450 monooxygenase (CYP450) gene candidates from G. lucidum, which may be responsible for the GA biosynthesis from lanosterol but have not been functionally characterized. As a result, overexpression of a CYP450 gene cyp5150l8 was firstly found to produce an antitumor GA, 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) in S. cerevisiae, as confirmed by HPLC, LC-MS and NMR. A final titer of 14.5 mg/L of HLDOA was obtained at 120 hr of the yeast fermentation. Furthermore, our in vitro enzymatic experiments indicate that CYP5150L8 catalyzes a three-step biotransformation of lanosterol at C-26 to synthesize HLDOA. To our knowledge, this is the first report on the heterologous biosynthesis of GAs. The results will be helpful to the GA biosynthetic pathway elucidation and to future optimization of heterologous cell factories for GA production.

HSP70-Hrd1 axis precludes the oncorepressor potential of N-terminal misfolded Blimp-1s in lymphoma cells.

B lymphocyte-induced maturation protein-1 (Blimp-1) ensures B-cell differentiation into the plasma cell stage, and its instability constitutes a crucial oncogenic element in certain aggressive cases of activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). However, the underlying degradation mechanisms and their possible therapeutic relevance remain unexplored. Here, we show that N-terminal misfolding mutations in ABC-DLBCL render Blimp-1 protein susceptible to proteasome-mediated degradation but spare its transcription-regulating activity. Mechanistically, whereas wild-type Blimp-1 metabolism is triggered in the nucleus through PML-mediated sumoylation, the degradation of lymphoma-associated mutants is accelerated by subversion of this pathway to Hrd1-mediated cytoplasmic sequestration and ubiquitination. Screening experiments identifies the heat shock protein 70 (HSP70) that selects Blimp-1 mutants for Hrd1 association, and HSP70 inhibition restores their nuclear accumulation and oncorepressor activities without disrupting normal B-cell maturation. Therefore, HSP70-Hrd1 axis represents a potential therapeutic target for restoring the oncorepressor activity of unstable lymphoma-associated Blimp-1 mutants.The transcriptional repressor Blimp-1 has an important role in B-cell differentiation. Here the authors show that lymphoma-associated Blimp-1 mutants are selectively recognized by HSP70-Hrd1, which leads to their accelerated degradation and propose HSP70 inhibition as a therapeutic approach for certain lymphomas.

Evolution of saturated hydraulic conductivity with compression and degradation for municipal solid waste.

Municipal solid waste (MSW) specimens were created from synthetic fresh MSW degraded in a laboratory scale enhanced degradation reactor. The degree of degradation and saturated hydraulic conductivity ks were measured to study the effects of compression and degradation on ks of MSW. The degree of degradation was characterized through the ratio of cellulose content to lignin content (i.e., C/L) and the loss ratio of volatile solid (i.e., DOD). ks of MSW specimens with different degrees of degradation was measured through triaxial permeameter tests under different confining pressures. It was found that, when the degradation time increased from 0month to 18months, ks decreased less than 1 order of magnitude for specimens with the same porosity (i.e., n=0.63 or 0.69). However, for specimens with the same degradation time, the decrease of ks could reach 2 orders of magnitude with n decreasing from 0.8 to 0.6. It indicates that compression has much greater influence on the reduction of ks than that of degradation. Based on the Kozeny-Carman model and first-order kinetics, a prediction model related to n and C/L (or DOD) of MSW was proposed to analyze the evolution of ks with compression and biodegradation. The methods to determine the values of model parameters were also proposed.

Rosiglitazone elevates sensitization of drug-resistant oral epidermoid carcinoma cells to vincristine by G2/M-phase arrest, independent of PPAR-γ pathway.

Rosiglitazone (ROSI), an oral antidiabetic agent, has been reported the anti-cancer properties recent years. In this paper, the potency of ROSI as a synergistic drug for vincristine (VCR) on resistant oral cancer cells was investigated. We found that ROSI potently enhanced the susceptibility of KB cells or KB/V cells to VCR in a dose manner and the synergy in KB/V cells was much more prominent than that in KB cells. The synergistic anti-proliferative effect of ROSI and VCR was associated with inhibition on tubulin polymerization, cell cycle arrest in G2/M phase and cell apoptosis induction, but has no effect on drug efflux-protein P-gp and was independent with PPARγ. The combination treatment of ROSI and VCR could regulate the PTEN/PI3K/AKT survival pathway with an upregulation of PTEN and down-regulation of p-AKT. The effect of G2/M phase arrest was associated with the upregulation of cyclin B1 and downregulation of p-cdc2. The apoptosis induction of ROSI and VCR was partly due to an upregulation of cleaved PARP and downregulation of Bcl-2/Bax ratio. In addition, combination treatment of ROSI and VCR had also shown anti-angiogenic effect by suppressing the migration and blocking the capillary tube formation of HUVECs. More importantly, this combination treatment induced an acceptably weak cytotoxicity in human normal HL-7702 cells, GES-1 cells and HUVECs. Taken together, ROSI may be used as a potential compound for combinatorial therapy or as a complement to VCR for treatment on oral cancer, especially on that have acquired resistance to VCR therapy.

Reactive oxygen species mediate oridonin-induced apoptosis through DNA damage response and activation of JNK pathway in diffuse large B cell lymphoma.

This study investigated the cytotoxic effect of oridonin (ORI), a diterpenoid isolated from Rabdosia rubescens, in human diffuse large B cell lymphoma (DLBCL) in vitro and in vivo and the potential molecular mechanisms for ORI-induced cell apoptosis. ORI treatment caused reactive oxygen species (ROS)-mediated oxidative DNA damage response (DDR) and the c-Jun N-terminal kinase (JNK) pathway activation, leading to an induction of intrinsic apoptosis. ROS abolition blocked ORI-induced apoptosis and attenuated the expression of phospho-histone H2AX and phospho-JNK, indicating that ROS-mediated DNA damage and JNK pathway activation were involved in ORI-induced apoptosis. The systemic administration of ORI suppressed the growth of human DLBCL xenografts without showing significant toxicity. These findings suggest that ORI may have promising therapeutic application in DLBCL.

Combination of rituximab and mammalian target of rapamycin inhibitor everolimus (RAD001) in diffuse large B-cell lymphoma.

Abstract We evaluated the efficacy of the anti-CD20 monoclonal antibody rituximab in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus for treating diffuse large B-cell lymphoma (DLBCL). The combination of rituximab and everolimus was more effective for inhibiting cell growth compared with single-agent therapy. An increase in G0/G1 cell cycle arrest and an increased population of cells in apoptosis were observed in the combination treatment group. The addition of rituximab reduced the overexpression of p-AKT caused by the negative feedback loop of everolimus and had an enhanced effect on inhibition of mTOR signaling, thus providing a rationale for this synergistic effect. Furthermore, combination treatment was also more effective than treatment with either agent alone for inhibiting the growth of DLBCL xenografts. Our study provides preclinical evidence and a theoretical basis for combination therapy with rituximab and everolimus in DLBCL.

A tetramethylpyrazine piperazine derivate CXC137 prevents cell injury in SH-SY5Y cells and improves memory dysfunction of rats with vascular Dementia.

We investigated the effects of CXC137, a tetramethylpyrazine piperazine derivate, on cell damage induced by N-methyl-D-aspartate (NMDA) in human derived neuroblastoma cells (SH-SY5Y) and its effect on memory dysfunction of rats with vascular dementia. It was found that the presence of CXC137 increased SH-SY5Y cells viability by inhibition of cell apoptosis induced by NMDA. These effects of CXC137 were accompanied by increases of the antioxidant superoxide dismutase activity and the level of reduced glutathione, and a decrease of lipid peroxidation product, malondialdehyde. The presence of CXC137 also showed to produce strong inhibition of cellular lactate dehydrogenase leakage, cell apoptosis and intracellular calcium overload. In a vascular dementia rat model established by bilateral common carotid arteries occlusion, treatment with CXC137 from 2 to 35 day of post-operation significantly improves the motor performance, spatial learning and memory capability of rats in both the prehensile traction test and Morris water maze test, an effect that was companied by reductions of the animal glutamic acid levels and the degree of brain mitochondrial swelling. These results suggest that CXC137 can improve the memory dysfunction in dementia and thus has important therapeutic potential for the treatment of dementia.

Activation of the PI3K/AKT/mTOR pathway in diffuse large B cell lymphoma: clinical significance and inhibitory effect of rituximab.

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma and accounts for approximately 30% of newly diagnosed lymphoid neoplasms in Western countries, and 40-50% in China. A better understanding of the biology of DLBCL is needed for the development of potential therapeutic agents that target specific intracellular pathways. In this study, expression of the important components of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway and their clinical significance were investigated in 73 DLBCL cases. The effect of rituximab alone or combined with the PI3K/AKT/mTOR pathway inhibitor rapamycin was further evaluated in the DLBCL cell lines. A total of 73 patients were identified, including 45 men and 28 women aged 18 to 78 years (median age 50 years). Of these patients, p-AKT was positive in 40 cases (54.8%), p-p70S6K in 34 cases (46.6%), and p-4E-BP1 in 33 cases (45.2%). Activation of the PI3K/AKT/mTOR pathway was related to poor disease outcome in DLBCL patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) but not in those treated with rituximab-CHOP. Rituximab combined with rapamycin synergically downregulated the PI3K/AKT/mTOR signaling pathway. Western blot analysis revealed a baseline activation status of the PI3K/AKT/mTOR pathway in DLBCL cell lines, with high levels of p-AKT, p-mTOR, in addition to downstream molecules p-p70S6K and p-4E-BP1. The results indicate that the PI3K/AKT/mTOR pathway is a potentially important signaling route and an unfavorable prognostic factor for DLBCL. Patients with PI3K/AKT/mTOR activation experience a more rapidly deteriorating clinical course with poor treatment response and decreased survival time. Addition of rituximab could downregulate PI3K/AKT/mTOR activation, reversing its negative effect on chemotherapy-treated patients. In addition, our results indicate that the combination of rituximab and inhibition of the activated PI3K/AKT/mTOR pathway could be a promising target for DLBCL therapeutic intervention in the future.