Establishment of a risk model correlated with metabolism based on RNA-binding proteins associated with cell pyroptosis in acute myeloid leukemia.

Background: RNA-binding protein (RBP) regulates acute myeloid leukemia (AML) by participating in mRNA editing and modification. Pyroptosis also plays an immunomodulatory function in AML. Therefore, this study aimed to identify pyroptosis-related RBP genes that could predict the prognosis of AML patients. Methods: AML related expression data were downloaded from the UCSC website and Gene Expression Omnibus (GEO) database. Pyroptosis-RPB-related differentially expressed genes (PRBP-DEGs) were conducted with a protein-protein interactions (PPI) network to screen out the key PRBP-DEGs, based on which a risk model was constructed by Cox analysis, and evaluated by plotting Receiver operating characteristic (ROC) curves and survival curves. Independent prognostic analysis was performed and a nomogram was constructed. Finally, enrichment analysis was performed for high and low risk groups. Reuslts: A total of 71 PRBP-DEGs were obtained and a pyroptosis-RPB-related risk model was constructed based on IFIT5, MRPL14, MRPL21, MRPL39, MVP, and PUSL1 acquired from Cox analysis. RiskScore, age, and cytogenetics risk category were identified as independent prognostic factors, and the nomogram based on these independent prognostic factors could accurately predict 1-, 3- and 5-year survival of AML patients. Gene set enrichment analysis (GSEA) showed that the high-risk and low-risk groups were mainly enriched in metabolic- and immune-related processes and pathways. Conclusion: In this study, a risk score model correlated with metabolism based on RNA-binding proteins associated with cell pyroptosis in acute myeloid leukemia was established, which provided a theoretical basis and reference value for therapeutic studies and prognosis of AML.

Antithymocyte Globulin for Matched Sibling Donor Transplantation in Patients With Hematologic Malignancies: A Multicenter, Open-Label, Randomized Controlled Study.

PURPOSE: The role of antithymocyte globulin (ATG) in preventing acute graft-versus-host disease (aGVHD) after HLA-matched sibling donor transplantation (MSDT) is still controversial. PATIENTS AND METHODS: We performed a prospective, multicenter, open-label, randomized controlled trial (RCT) across 23 transplantation centers in China. Patients ages 40-60 years with standard-risk hematologic malignancies with an HLA-matched sibling donor were randomly assigned to an ATG group (4.5 mg/kg thymoglobulin plus cyclosporine [CsA], methotrexate [MTX], and mycophenolate mofetil [MMF]) and a control group (CsA, MTX, and MMF). The primary end point of this study was grade 2-4 aGVHD on day 100. RESULTS: From November 2013 to April 2018, 263 patients were enrolled. The cumulative incidence rate of grade 2-4 aGVHD was significantly reduced in the ATG group (13.7%; 95% CI, 13.5% to 13.9%) compared with the control group (27.0%; 95% CI, 26.7% to 27.3%; P = .007). The ATG group had significantly lower incidences of 2-year overall chronic GVHD (27.9% [95% CI, 27.6% to 28.2%] v 52.5% [95% CI, 52.1% to 52.9%]; P < .001) and 2-year extensive chronic GVHD (8.5% [95% CI, 8.4% to 8.6%] v 23.2% [95% CI, 22.9% to 23.5%]; P = .029) than the control group. There were no differences between the ATG and control groups with regard to cytomegalovirus reactivation, Epstein-Barr virus reactivation, 3-year nonrelapse mortality (NRM), 3-year cumulative incidence of relapse (CIR), 3-year overall survival, or 3-year leukemia-free survival. Three-year GVHD relapse-free survival was significantly improved in the ATG group (38.7%; 95% CI, 29.9% to 47.5%) compared with the control group (24.5%; 95% CI, 16.9% to 32.1%; P = .003). CONCLUSION: Our study is the first prospective RCT in our knowledge to demonstrate that ATG can effectively decrease the incidence of aGVHD after MSDT in the CsA era without affecting the CIR or NRM.

Chronic myeloid leukemia following liver transplantation: A case report.

Long-term utilization of immunosuppression in organ transplant recipients leads to decreased immune-mediated tumor surveillance and increased risk of developing malignant tumors. However, chronic myeloid leukemia (CML) following living donor liver transplantation (LDLT) is rarely reported. The current case report presents a 42-year-old male patient who developed CML 14 months following LDLT. The patient achieved complete hematologic remission and early molecular response at 3 months imatinib treatment and major molecular response at 12 months imatinib treatment. The pathogenesis, risk factors, treatment and prognosis for CML following liver transplantation are unclear. Therefore, further analysis through accumulation of cases will be of great importance to prevent and treat this rare complication following liver transplantation.

PIM-1 kinase inhibitor SMI-4a exerts antitumor effects in chronic myeloid leukemia cells by enhancing the activity of glycogen synthase kinase 3ß.

The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus­1 (PIM­1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI­4a is a selective PIM­1 kinase inhibitor that inhibits PIM­1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI­4a in K562 and imatinib­resistant K562 (K562/G) cell lines. It was demonstrated that SMI­4a inhibited the proliferation of K562 and K562/G cells using a WST­8 assay. The Annexin V­propidium iodide assay demonstrated that SMI­4a induced apoptosis of K562 and K562/G cells in a dose­, and time­dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI­4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI­4a decreased phosphorylated (p)­Ser9­glycogen synthase kinase (GSK) 3ß/pGSK3ß and inhibited the translocation of ß­catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP­ribose) polymerase­1 was upregulated, and apoptosis regulator Bcl­2 and Myc proto­oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of ß­catenin in the plasma and nucleus. The results of the present study suggest that SMI­4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.

Allogeneic Bone Marrow-Derived Mesenchymal Stromal Cells Expanded In Vitro for Treatment of Aplastic Anemia: A Multicenter Phase II Trial.

We conducted a phase II, noncomparative, multicenter study to assess the efficacy and safety of allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) expanded in vitro for patients with aplastic anemia (AA) refractory to immunosuppressive therapy. Seventy-four patients from seven centers received allogeneic BM-MSCs at a dose of 1-2 × 106 cells/kg per week for 4 weeks. Responses were assessed at 0.5, 1, 2, 3, 6, 9, and 12 months after the first cells infusion. Patients with response at 1 month continued to receive four infusions. All patients were evaluable. The overall response rate was 28.4% (95% confidence interval, 19%-40%), with 6.8% complete response and 21.6% partial response. The median times to response of leukocytic, erythrocytic, and megakaryocytic linages were 19 (range, 11-29), 17 (range, 12-25), and 31 (range, 26-84) days, respectively. After median follow-up of 17 months, overall survival was 87.8%. Seven patients developed transitory and mild headache and fever, but no other adverse events were observed. Antithymocyte globulin used in previous treatment and no activated infection throughout treatment were predictors for response. Allogeneic BM-MSCs infusion is a feasible and effective treatment option for refractory AA. The trial was registered at www.clinicaltrials.gov as NCT00195624. Stem Cells Translational Medicine 2017;6:1569-1575.

Tuberculosis-associated hemophagocytic lymphohistiocytosis in an umbilical cord blood transplant recipient.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal condition that can be primary or secondary. Secondary HLH due to Mycobacterium tuberculosis (TB) is uncommon. We report a case of tuberculosis-associated HLH in an umbilical cord blood transplant (UCBT) recipient and discuss its clinical characteristics and challenges. METHODS: Hematologic investigations, bone marrow aspirates, Xpert MTB/RIF test of TB with peripheral blood were performed. Immune modulation with anti-TB therapy was initiated. RESULTS: Subsequent treatment with anti-TB treatment resulted rapid clinical response and disease remission. CONCLUSION: It is important to consider TB as one of the underlying cause of HLH in high-risk patients, particularly those in immunodeficient states. Early diagnosis and treatment can improve the survival rates of patients with tuberculosis-associated HLH.

Aurora A Kinase Inhibitor AKI603 Induces Cellular Senescence in Chronic Myeloid Leukemia Cells Harboring T315I Mutation.

The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.

Zoledronic acid overcomes adriamycin resistance in acute myeloid leukemia cells by promoting apoptosis.

Zoledronic acid (ZOL), a nitrogen­containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti­leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti­leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL­60 and adriamycin­resistant HL­60 (HL­60/A) cells using a WST­8 assay. An Annexin V­propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose­ and time­dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S­phase cell cycle arrest, downregulation of B­cell lymphoma 2 (Bcl­2) and upregulation of Bcl­2 associated X protein and cleaved poly (ADP­ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti­leukemic activity appeared notably enhanced in HL­60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.

Celecoxib suppresses autophagy and enhances cytotoxicity of imatinib in imatinib-resistant chronic myeloid leukemia cells.

BACKGROUND: Chronic myelogenous leukemia (CML) is a hematological stem cell disorder. Tyrosine kinase inhibitors (TKIs) are the standard treatments for CML, but a number of patients fail to respond effectively due to gene mutations. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, has been shown to have anti-tumor effect on solid tumor whereas the anti-CML effect and its underlying mechanism have not been completely elucidated. METHODS: The cytotoxic effects of celecoxib and/or imatinib were evaluated by MTT assay. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis or necrosis was analyzed by Annexin-V/PI, Hoechst 33342 staining and Western blot assays. Autophagy suppression effect of celecoxib was examined by Western blot and LysoTracker probe labelling. Lysosensor probe labelling was used to detect the effect of celecoxib on the lysosomal function. RESULTS: In this study, we found that celecoxib had therapy efficacy in KBM5 and imatinib-resistant KBM5-T315I CML cell lines. Celecoxib caused significant cytotoxic effect in both cell lines, especially in KBM5-T315I cells exposed to celecoxib for 72 h. Moreover, celecoxib induced necrosis and apoptosis while inhibited autophagy in CML cell lines and patient samples. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function. Celecoxib was tested in combination with imatinib, demonstrating that celecoxib could strengthen the cytotoxicity of imatinib in imatinib-resistant CML cells. CONCLUSIONS: These findings showed that celecoxib had therapy efficacy on CML cells. And it is first time to demonstrate that celecoxib is an autophagy suppresser and a combination of celecoxib and imatinib might be a promising new therapeutic strategy for imatinib-resistant CML cells.

RUNX3 plays an important role in As2O3­induced apoptosis and allows cells to overcome MSC­mediated drug resistance.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt­related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)­mediated protection of leukemia cells from As2O3­induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3­induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3­induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3­induced cellular responses and allows cells to overcome MSC­mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC­mediated drug resistance.

Targeting GLI1 Suppresses Cell Growth and Enhances Chemosensitivity in CD34+ Enriched Acute Myeloid Leukemia Progenitor Cells.

BACKGROUND/AIMS: Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61. METHODS: The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61. RESULTS: Kasumi-1 and KG1a cells, containing more CD34+ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34+ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34+ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34+ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity. CONCLUSIONS: The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

[Expression of Gli1 in Adult Acute Lymphoblastic Leukemia and Its Clinical Significance].

OBJECTIVE: To explore the expression and clinical significance of Hedgehog signaling transcription factor Gli1 in acute lymphoblastic leukemia (ALL) patients. METHODS: The clinical specimens were obtained from 32 newly diagnosed and 6 relapsed ALL patients. Normal bone marrow cells from 15 healthy donors were used as controls. Real-time qPCR and Western blot were applied to detect Gli1 mRNA and protein expression in bone marrow mononuclear cells (BMMNC) of these samples respectively. The relation of Gli1 mRNA levels with clinical parameter was also evaluated. RESULTS: The expression level of Gli1 mRNA in de novo and relapsed ALL patients was significantly higher than that in the normal controls (P < 0.05). There was no stalistically significant difference of the Gli1 mRNA expression between de novo and relapsed ALL cases (P > 0.05). In 24 de novo ALL patients with complete remission (CR) after induction chemotherapy, the levels of Gli1 mRNA were significantly reduced as compared with levels before treatment (P < 0.05). However, in 4 ALL patients without remission, no obvious difference of Gli1 mRNA levels were observed as compared with levels of Gli1 before treatment (P > 0.05). A positive correlation between the Gli1 mRNA expression level and white blood cell count (WBC) was found in the BMMNC of ALL patients (R = 0.725, P < 0.05). Similarly, Gli1 protein expression was significantly higher in the de novo and relapsed ALL cases compared with normal controls. The Gli1 protein level was down-regulated when the ALL patients was in CR. CONCLUSION: The expression of Gli1 mRNA and protein has been found to be high in de novo and relapsed ALL patients, and the change of Gli1 expression maybe relate to therapeutic efficacy and prognosis of ALL patients.

A novel compound against oncogenic Aurora kinase A overcomes imatinib resistance in chronic myeloid leukemia cells.

Drug resistance still represents a major obstacle to successful chronic myeloid leukemia (CML) treatment and novel compounds or strategies to override this challenging problem are urgently required. Here, we evaluated a novel compound AKI603 against oncogenic Aurora kinase A (Aur-A) in imatinib-resistant CML cells. We found that Aur-A was highly activated in imatinib-resistant KBM5-T315I cells. AKI603 significantly inhibited the phosphorylation of Aur-A kinase at Thr288, while had little inhibitory effect on BCR-ABL kinase in both KBM5 and KBM5-T315I cells. AKI603 inhibited cell viability, and induced cell cycle arrest with polyploidy accumulation in KBM5 and KBM5-T315I cells. Moreover, inhibition of Aur-A kinase by AKI603 suppressed colony formation capacity without promoting obvious apoptosis. Importantly, AKI603 promoted cell differentiation in both CML cell types. Thus, our study suggested the potential clinical use of small molecule Aurora kinase inhibitor AKI603 to overcome imatinib resistance in CML treatment.

Inhibition of c-Myc overcomes cytotoxic drug resistance in acute myeloid leukemia cells by promoting differentiation.

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPß contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPß could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPß. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPß in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.

ATO/ATRA/anthracycline-chemotherapy sequential consolidation achieves long-term efficacy in primary acute promyelocytic leukemia.

The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3, ATO) has been effective in obtaining high clinical complete remission (CR) rates in acute promyelocytic leukemia (APL), but the long-term efficacy and safety among newly diagnosed APL patients are unclear. In this retrospective study, total 45 newly diagnosed APL patients received ATRA/chemotherapy combination regimen to induce remission. Among them, 43 patients (95.6%) achieved complete remission (CR) after induction therapy, followed by ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment with a median follow-up of 55 months. In these patients, the estimated overall survival (OS) and the relapse-free survival (RFS) were 94.4% ± 3.9% and 94.6 ± 3.7%, respectively. The toxicity profile was mild and reversible. No secondary carcinoma was observed. These results demonstrated the high efficacy and minimal toxicity of ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment for newly diagnosed APL in long-term follow-up, suggesting a potential frontline therapy for APL.

Acute pleural and pericardial effusion induced by chemotherapy in treating chronic myelocytic leukemia.

BACKGROUND: Report of a rare and serious complication of chemotherapy with daunorubicin and cytarabine (DA regimen). METHODS: We report a special case of a patient diagnosed chronic myeloid leukemia (CML) with accelerated phase, who simultaneously suffered from acute pleural and pericardial effusion while receiving chemotherapeutic treatment with DA regimen. RESULTS: Following treatment with DA regimen, the patient had the symptoms of chest distress and shortness of breath, followed by respiratory failure and pericardial tamponade. The patient's condition was improved when treated with the puncturation through the pericardium and pleural cavity, coupled with glucocorticosteroid treatment. CONCLUSIONS: Physicians should be made aware of the potential for emergency pleural and pericardial effusion caused by daunorubicin and cytarabine in order to accurately diagnose and treat these conditions and thereby decrease mortality related to chemotherapy.

Diagnosis of Felty's syndrome, distinguished from hematological neoplasm: A case report.

Felty's syndrome (FS) is characterized by the three conditions of rheumatoid arthritis (RA), neutropenia and splenomegaly, and occurs in few cases of longstanding erosive RA. Discriminating between rare occurrences of autoimmune diseases and malignancies is crucial. The present study describes the case of a 17-year-old female with a two-year history of RA, presenting with an irregular fever, hepatosplenomegaly and enlarged lymph nodes. The antinuclear antibody titer was 1:320, while antibody results for anti-dsDNA, anti-Sm and rheumatoid factor were negative. The clinical presentation was similar to that of lymphoma. However, the fluorodeoxyglucose-positron emission tomography and biopsy examinations of the liver and cervical lymph node did not support the diagnosis of lymphoma. According to the laboratory results and clinical symptoms, the differential diagnosis indicated FS, and immunosuppressive agents were administered. Two weeks later, the patient no longer had a fever, and the transaminase levels were normal, associated with shrinkage of the liver and spleen.

Inhibition of autophagy augments the anticancer activity of α-mangostin in chronic myeloid leukemia cells.

Natural products possessing anticancer activity have been extensively studied because of their low toxicity and potential effect. α-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor properties. This study was carried out to investigate how to improve the anticancer effects of α-mangostin in chronic myeloid leukemia (CML) cell lines bearing wild-type BCR-ABL or BCR-ABL-T315I mutation. We showed that α-mangostin inhibited cell proliferation of K562, KBM5 and KBM5-T315I cells in both a time- and dose-dependent manner. Significantly, α-mangostin increased the number of apoptotic cells and induced DNA fragmentation compared to control cells. Moreover, α-mangostin selectively inhibited proliferation in primary CML cells, while showing limited lethality in normal hematopoietic progenitors. Additionally, α-mangostin induced not only apoptosis but also autophagy in CML cells. α-Mangostin dramatically increased the expression levels of LC-3II, an autophagosome marker in mammals, and the accumulation of autophagic vacuoles (AVs). Inhibition of autophagy by chloroquine enhanced α-mangostin-mediated cytotoxicity through increasing apoptosis. Taken together, our data suggest that targeting the autophagy pathway is a promising therapeutic strategy to enhance α-mangostin-induced apoptosis. Our study provides an approach for future studies to explore this combination for the treatment of CML.

Antibody-directed double suicide gene therapy targeting of MUC1- positive leukemia cells in vitro and in vivo.

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.