A novel approach for relapsed/refractory FLT3mut+ acute myeloid leukaemia: synergistic effect of the combination of bispecific FLT3scFv/NKG2D-CAR T cells and gilteritinib.

BACKGROUND: Patients with relapsed/refractory acute myeloid leukaemia (AML) with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) have limited treatment options and poor prognosis. Therefore, novel treatment modalities are needed. Since high expression of natural killer group 2 member D ligands (NKG2DLs) can be induced by FLT3 inhibitors, we constructed dual-target FLT3 single-chain fragment variable (scFv)/NKG2D-chimeric antigen receptor (CAR) T cells, and explored whether FLT3 inhibitors combined with FLT3scFv/NKG2D-CAR T cells could have synergistic anti-leukaemia effects. METHODS: FLT3scFv and NKG2D expression in CAR T cells, FLT3 and NKG2DL expression in AML cells, and the in vitro cytotoxicity of combining CAR T cells with gilteritinib were assessed by flow cytometry. The therapeutic effect was evaluated in a xenograft mouse model established by injection of MOLM-13 cells. Mechanisms underlying the gilteritinib-induced NKG2DL upregulation were investigated using siRNA, ChIP-QPCR and luciferase assays. RESULTS: The FLT3scFv/NKG2D-CAR T cells specifically lysed AML cells both in vitro and in the xenograft mouse model. The efficacy of FLT3scFv/NKG2D-CAR T cells was improved by gilteritinib-pretreatment. The noncanonical NF-κB2/Rel B signalling pathway was found to mediate gilteritinib-induced NKG2DL upregulation in AML cells. CONCLUSIONS: Bispecific FLT3scFv/NKG2D-CAR T cells can effectively eradicate AML cells. The FLT3 inhibitor gilteritinib can synergistically improve this effect by upregulating NF-κB2-dependent NKG2DL expression in AML cells.

Real-time quantitative polymerase chain reaction assay for detecting the eps8 gene in acute myeloid leukemia.

BACKGROUND: Eps8 is a novel proto-oncogene related to the tumorigenesis, proliferation, metastasis, chemo-resistance, and prognosis of many human solid cancers. However, the function of Eps8 in acute myeloid leukemia (AML) is still unclear. Thus, this study aims to develop a real-time quantitative polymerase chain reaction (PCR) assay for Eps8 in AML. METHODS: Eps8 was amplified and cloned into pMD18-T to generate the recombinant plasmid pMD18-T/Eps8 as standard DNA for the establishment of real-time quantitative PCR. This assay was used to detect the expression level of Eps8 in bone marrow samples from AML patients and healthy volunteers (control group). RESULTS: The limit of detection achieved using this standard was 100 copies, which was 10 times more sensitive than that achieved using conventional PCR, indicating high sensitivity. Melting curve analysis demonstrated that the primers designed for the established real-time quantitative PCR assay were specific and available. The expression level of Eps8 in the AML patients increased compared with that in the control group (p = 0.013). Furthermore, the expression level of Eps8 showed significant correlation with the complete remission rate of AML patients to chemotherapy (p = 0.024). CONCLUSIONS: The established assay is useful in detecting the expression level of Eps8. The results suggest that Eps8 may serve as a prognostic factor of responsiveness to chemotherapy in AML patients.

Venetoclax enhances NK cell killing sensitivity of AML cells through the NKG2D/NKG2DL activation pathway.

BACKGROUND: Venetoclax, a selective B-cell lymphoma-2 (BCL2) inhibitor, has a potential therapeutic effect when combined with demethylating agents in the first-line setting of unfit elderly patients with acute myeloid leukaemia (AML); however, efficacy is still limited in refractory/recurrent AML. Therefore, exploration of a suitable novel treatment scheme is urgently needed.However, combining venetoclax with NK cell-based immunotherapy has not been studied. METHODS: The cytotoxicity of NK cell combined with venetoclax was assessed in vitro using flow cytometry. Venetoclax-induced natural killer group 2 member D (NKG2D) ligand (NKG2DL) expression was detected by flow cytometry and western blotting. Mechanisms underlying venetoclax-induced NKG2DL expression were found by GSE127200 analysis and investigated using real-time PCR (Q-PCR) and western blotting. RESULTS: Flow cytometric analysis showed that combining venetoclax with NK cells produced synergistic anti-leukaemia effects similar to those of venetoclax + azacitidine. Venetoclax could render AML cell lines and primary AML cells sensitive to NK cell killing by promoting NK cell degranulation, NK-AML cell recognition and NK cell secretion of interferon (IFN)-γ and granzyme B. The synergistic effect resulted from venetoclax-induced NKG2DL upregulation in AML cells and could be undermined by blocking NKG2D on NK cells. This finding suggests that venetoclax enhances NK cell killing activity by activating the NKG2D/NKG2DL ligand-receptor pathway. Furthermore, the nuclear factor-kappa-B (NFKB) signalling pathway was involved in venetoclax-induced NKG2DL upregulation. CONCLUSIONS: Collectively, our data confirm that venetoclax combined with NK cells induces synergistic AML cell cytolysis and preliminarily revealed that venetoclax could selectively induce NKG2DLs on AML cells via NFKB signalling pathway.

NeoPeptide: an immunoinformatic database of T-cell-defined neoantigens.

Therapeutic vaccines represent a promising immunotherapeutic modality against cancer. Discovery and validation of antigens is the key to develop effective anti-cancer vaccines. Neoantigens, arising from somatic mutations in individual cancers, are considered as ideal cancer vaccine targets because of their immunogenicity and lack of expression in normal tissues. However, only few databases support convenient access to these neoantigens for use in vaccines. To address this gap, we developed a web-accessible database, called NeoPeptide, which contains most of the important characteristics of neoantigens (such as mutation site, subunit sequence, major histocompatibility complex restriction) derived from published literature and other immunological resources. NeoPeptide also provides links to resources for further characterization of the novel features of these neoantigens. NeoPeptide will be regularly updated with newly identified and published neoantigens. Our work will help researchers in identifying neoantigens in different cancers and hasten the search for appropriate cancer vaccine candidates.

[Effect of Silencing Eps8 Gene Expression on the Biology Activity of Human Leukemia K562 Cells and Its Molecular Mechanism].

OBJECTIVE: The study was to investigate the effect of silencing Eps8 gene expression on proliferation and apoptosis of human leukemia K562 cells and its molecular mechanism. METHODS: The expetriments were divided into 3 groups, including blank control group(K562 cells without treatment), K562-shRNA group(K562 cells transfected by specific Eps8-shRNA lenticiral vector) and K562-NC group(K562 cells transfected by negtive control lenticiral vector). K562 cells with stably-silenced Eps8 gene were constucted by lentibirus-mediated RNA technology. The efficacy of transfection was observed by fluorescence microscopy and the changes of Eps8 mRNA and protein level were detected by RT-PCR and Western blot respectively. Cell proliferation was confirmed by typan blue exclusion and MTT method. The apoptosis rate of cells was analysed by the flow cytometry, and colony forming was detected by methylcellulose colony forming assay. The protein level change of phosphrylated-AKT were detected by Western blot. RESULTS: Stably-silenced Eps8 gene K562 cells and the negative control cells were successfully constructed. Compared with the blank control group and the K562-NC group, the proliferation of K562-shRNA cells were siginificantly inhibited(P<0.05); the apoptosis of K562-shRNA cells increased(P<0.05). In addition, the methylcellulose colony forming assay showed that the colony forming was dramatically suppressed in K562-shRNA cells (P<0.05). Furthermore, knocking down Eps8 gene reduced the protein level of AKT phosphrylation at both residue Ser437 and Thr308(P<0.05), while there was no obvious change in the level of total-AKT(P>0.05). Knocking down Eps8 gene reduced the protein level of m-TOR phosphrylation and PRAS40 phosphrylation (P<0.05), while there was no obvious change in the level of total-mTOR and PRAS40 (P>0.05). CONCLUSION: Silencing Eps8 gene through lentvirus can inhibit the cell proliferation and promote the apoptosis of human leukemia K562 cells, which possibly relates with the inhibition of AKT/mTOR activation.

Sunitinib Induces NK-κB-dependent NKG2D Ligand Expression in Nasopharyngeal Carcinoma and Hepatoma Cells.

Multitargeted tyrosine kinase inhibitors (MTKIs) have been shown to combine with natural killer (NK) cell adoptive transfer for the treatment in various cancers. MTKIs sensitize cancer cells to NK cell therapy through upregulation of nature killer group 2 member D ligands (NKG2DLs) on tumor cells. However, the molecular mechanism of MTKIs-mediated upregulation of NKG2DLs is still unknown. In this study, we confirmed sunitinib induced downregulation of its targets, such as vascular endothelial growth factor, platelet-derived growth factor, and c-kit in multiple-drug-resistant nasopharyngeal carcinoma cell line CNE2/DDP and hepatoma cell line HepG2. Then, we further showed sunitinib induced cell proliferation inhibition, apoptosis, and DNA damage in CNE2/DDP and HepG2 cells. Coculture experiments showed that sunitinib-treated CNE2/DDP and HepG2 cells were able to increase the activation and cytotoxicity of NK cells. Quantitative polymerase chain reaction results showed that sunitinib upregulated NKG2DLs, apoptotic genes, DNA damage repair genes, and nuclear factor (NF)-κß family genes. Silencing of NF-κß1, NF-κß2, or RelB (NF-κß pathway) inhibited sunitinib-induced upregulation of NKG2DLs. Taken together, we concluded that sunitinib upregulated NKG2DLs through NF-κß signaling noncanonical pathway which might mediate higher cytotoxic sensitivity of CNE2/DDP and HepG2 cells to NK cells.

[Prediction and Identification of HLA-A*0201 Restricted CTL Epitopes from Eps8].

OBJECTIVE: To find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic. METHODS: Online biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice. RESULTS: Four natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner. CONCLUSION: Natural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.

[ApoE4 increases glycogen synthase kinase 3ß expression and Tau phosphorylation in U87 cells].

OBJECTIVE: To explore the relations among apolipoprotein E4, Tau protein and glycogen synthase kinase 3ß (GSK-3ß). METHODS: U87 cells were transfected with pIRES-EGFP (control) or the recombinant plasmids ApoE4/pIRES-EGFP or ApoE3/pIRES-EGFP, and the expression levels of p-Tau/Tau and GSK-3ß in the cells were examined with Western blotting. To further confirm the effect of ApoE on GSK-3ß and p-Tau expressions, a short interfering RNA (siRNA) targeting ApoE (ApoE-siRNA) was transfected into U87 cells via Lipofectamine 2000 and the protein expressions were examined 24 h later. RESULTS: Compared with those in the control group, the expressions levels of both GSK-3ß and p-Tau/Tau increased significantly in the cells transfected with ApoE4 and ApoE3 plasmids (P<0.01), and the ApoE4 plasmid produced a more potent effect than the ApoE3 plasmid on the protein expressions (P<0.01). ApoE knockdown resulted in significantly reduced expressions of GSK-3ß (P<0.001) and p-Tau (P<0.01) in the cells. CONCLUSION: ApoE4 can enhance Tau phosphorylation though upregulating GSK-3ß, which sheds light on a new role of ApoE4 in Alzheimer's disease.

[Cytotoxicity of Naja Naja Actra Venom Component combined with activated immune cells on leukemia cell line KG1a].

This study was aimed to investigate the cytotoxic effect of the Naja Naja Actra Venom Component (NNAVC) combined with activated immune cells on human acute myeloblastic leukemia line KG1a cells. The cytotoxic effects of NNAVC at different concentrations on KG1a cells were measured by CCK-8 method. LDH releasing assay was used to detect the cytotoxic effects of activated immune cells, NNAVC combined with activated immune cells on KG1a cells and the sensitivity of KG1a treated with NNAVC to activated immune cells. The results showed that the inhibitory rate of NNAVC on KG1a cells increased with the concentration enhancement, the cytotoxicity of activated immune cells at the different effector to target (E:T) ratios(6.25:1, 12.5:1, 25:1) on KG1a cells were 12.30%, 24.85% and 52.26%. The cytotoxicity of NNAVC combined with activated immune cells at the different E:T cell ratios (10:1, 20: 1) on KG1a cells were 56.21% and 85.59%, which were higher than that of NNAVC or activated immune cells alone. The cytotoxicity of activated immune cells at the E: T cell ratio of 10:1 on KG1a cells treated with NNAVC at different concentrations were 25.65%, 31.33%, 28.63% and 16.78%, respectively, and that at the E:T cell ratio of 20: 1 were 40.62%, 44.70%, 44.62% and 40.72%. It is concluded that:both of NNAVC and activated immune cells have lethal effect on KG1a cells, and the combination of NNAVC and activated immune cells can strengthen their effect on KG1a.

Eps8 vaccine exerts prophylactic antitumor effects in a murine model: a novel vaccine for breast carcinoma.

Cancer vaccines are an effective way to prevent the occurrence of cancer. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel tumor-associated antigen, which is overexpressed in the majority of tumor types. In the present study, the Eps8 protein was cloned and characterized, and its feasibility as an antitumor agent in murine breast carcinoma was investigated. The results revealed that the Eps8 protein increased the secretion of interleukin (IL)-12 in the culture supernatant of dendritic cells (DCs). The Eps8 protein­pulsed DCs induced significant cytotoxic T lymphocyte (CTL) responses, T-cell proliferation and a higher level of interferon (IFN)-γ in the culture supernatant of the splenocytes ex vivo. Additionally, when the mice were immunized with the Eps8 vaccine, this resulted in a regression of 4T1 breast tumors and significantly prolonged survival time in the tumor­bearing mice compared with that in the phosphate-buffered saline (PBS) control group. The Eps8 vaccine induced higher CTL responses in the splenocytes of mice vaccinated against the 4T1 cells; the ratio of CD4+/CD8+ T cells was increased in the Eps8 group; and the percentage of CD4+CD25+ FoxP3+ regulatory T (Treg) cells in the Eps8 group was significantly lower compared with that of the PBS group. The results suggested that the Eps8 vaccine was able to stimulate antitumor effects against 4T1 breast cancer cells in vitro and in vivo, and it may provide a potential immunotherapeutic agent for the treatment of breast cancer.

[Advancement of peptide vaccines for hematologic malignancies].

Despite recent significant advances in the treatment of hematological malignancies, relapse of this disease is of great note with the existence of the minimal residual disease (MRD). Tumour peptide vaccine seems to be one of the effective immunotherapies for eliminating tumor cells of MRD. This review focuses on the late results of clinical trails of peptide vaccination protocols targeting WT1, RHAMM, BCR-ABL, PR1 in hematological malignancies and the development of specific immune responses to PRAME and Survivin peptides. An outlook to heteroclitic peptides, new adjuvants, combined peptide vaccines and Ad-tWT1 vaccine is also given to further explore the possibility to enhance the efficacy of the peptide vaccine.