1. Study On The Effect Of TBLR1-RARα On Leukemia And Its Therapeutic Strategy 2. Study On The Biological Function Of Acute Promyelocytic Leukemia Cells And Its Mechanism

Objective:TBLR1-RARα is a novel fusion gene of acute promyelocytic leukemia (APL) which was first discovered by our lab in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation. Our earlier work has clarified the characteristics of its basic structure and functions. In this study, we investigate the potential role of TBLR1-RARα fusion gene in the pathogenesis of APL in vivo at systematic biological level. Meanwhile we try to find the possible mechanisms by comparing the genes that expressed differentially between TBLR1-RARα mice and normal mice and new strategies to treat this kind of leukemia.Methods:To construct the MSCV-TBLR1-RARα-Flag-IRES-GFP retroviral plasmid (MSCV-TR plasmid), and transfect the 293T packaging cells with MSCV-TR plasmid to produce retroviruses, which were then used to transduce mouse bone marrow cells. PCR and Western blot assays were used to determine the expression level of TBLR1-RARα fusion gene. Lin- cells from mouse bone marrow were purified with lineage cell depletion kit and then infected with MSCV-TR and vehicle retroviral supernatant. For differentiation assay in vitro, the GFP+lin- cells were plated and incubated with or without all-trans retinoic acid(ATRA), then characterized by analyzing the myeloid markers (CD11b and GR-1). The proliferation ability was measured by colony formation assay. For the in vivo experiment,3-5×105 GFP+ lin-cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Numbers of GFP+ cells in peripheral blood as well as general condition of mice were monitored regularly.Wright’s staining and HE staining were used to assess the pathological types of leukemic mice. Cell surface markers were analyzed by FACS. The spleen cells from primary leukemia mice were inoculated to secondary recipient mice to test whether they could develop into leukemia. The colony forming ability of leukemia cells was studied using methylcellulose semi-solid medium. The response of leukemia cells to ATRA was examined by analyzing the expression of surface markers. Genome-wide expression profiling analysis was performed to identify differentially expressed genes between normal and TBLR1-RARαmice (TR mice). ATRA, arsenic trioxide (As2O3), chemotherapy drug Ara-C and histone deacetylase inhibitor (HDACi) NL-101 were used to explore better ways to treat TR mice.Results:The MSCV-TBLR1-RARa-Flag-IRES-GFP retroviral plasmid was successfully constructed. The expressing of TBLR1-RARagene was confirmed at mRNA and protein level. Colony formation assay showed that clonogenic potential of lin- cells expressing TBLR1-RARαwas enhanced. What’s more, the differentiation experiment indicated that TBLR1-RARαmay be prone to block the differentiation of hematopoietic stem cells at a relatively early stage. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARαexpressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. Many immature cells with high nucleus to cytoplasm ratios were observed in peripheral blood, bone marrow, spleen and liver by Wright-Giemsa staining. Histopathologic examination showed leukemic cells diffusely invading the bone marrow, spleen and liver. The phenotype analysis revealed that the progenitor markers CD34 and C-kit were positive, while Sca-1 was weakly positive, the myeloid lineage markers GR-1, Macl, CD16 were positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3, CD4 and CD8 were all negative. The mouse leukemia cells could be successfully transplanted in secondary and further recipients with shortened latency(3-4weeks), with C-kit+GR-1+ becoming the main phenotype of leukemia cells. Comparing with the normal mouse spleen cells, the TR mouse spleen cells had stronger colony forming ability. The phenotype and morphology analysis revealed that TR mouse cells were sensitive to ATRA treatment. Genome-wide expression profiling analysis showed that the differentially expressed genes were associated with cell development, proliferation, differentiation, apoptosis, migration and cell metabolism. These genes mainly involved in pathways in cancer, MAPK signaling pathway and metabolic pathways. Contrary to our expectation, TR mice treated with 1.5-2.5/kg ATRA alone or together with 2.0mg/kg As2O3 didn’t survive longer than control group, although the in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. But when treated with Ara-C or HDACi NL-101, the survival time was markedly longer than that of the control group.Conclusions:In conclusion, we successfully established the TBLR1-RARα leukemia mouse model and clarified its main characteristics. Meanwhile we identified lots of genes that might relate with the process of leukemogenesis. These results suggest that TBLR1-RARa plays a critical role in the leukemia genesis and development. This model system may represent a versatile tool to study the mechanisms of leukemogenesis and help to design new strategies for APL treatment.Objective:Chidamide is China’s first self-developed oral slective histone deacetylase inhibitor, which has been approved by the U.S. Food and Drug Administration for the treatment of peripheral T-Cell lymphoma (PTCL). In addition to treatment of PTCL, chidamide can also induce apoptosis in many other cancers, including lung cancer, breast cancer, prostate cancer, multiple myeloma and some AML cells. In this study, the anti-cancer effect of chidamide on acute promyelocytic leukemia cells and its mechanisms were investigated in vitro and in vivo.Methods:Cell viabilities of NB4 cells and the novel fusion gene TBLR1-RARa expressing U937 cells (U937-TR) were examined by methyl thiazolyl tetrazolium (MTT) assay and the half maximal inhibitory concentrations (IC50) of chidamide were also calculated. Flow cytometry was used to analyze the cell apoptosis rate, cell cycle distribution and cell differentiation rate. The expression changes of caspase3, PARP, CDK2 and CyclinEl after chidamide treatment were analyzed by Western blot. Flow cytometry and morphology analysis were used to detect the effect of chidamide on the apoptosis of APL patients’ cells and normal hematopoietic cells. For the experiments with TBLR1-RARa mouse model, colony forming assay was performed to detect the proliferating ability of mouse leukemia cells after drug treatment. The leukemic mice were randomized into four groups and received 0.1% sodium carboxyl methylcellulose and 5,12.5 or 25 mg/kg chidamide every two days by gavage for 20 days. Body mass and GFP+cells of peripheral blood were measured regularly. The survival time of every mouse was recorded and the differences between groups were compared.Results:MTT assay indicated that chidamide dramatically inhibited cell proliferation in both cell lines, their IC50 values at 48h were 0.068μM and 0.55μM respectively. Compared with U937-TR cells, NB4 cells seemed to more sensitive to chidamide. Chidamide triggered caspase-3 dependent apoptosis in dose-and time-dependent manner. Chidamide induced cell cycle arrest of NB4 and U937-TR cells in G0/G1 phase by downregulating the expression of CDK2 and CyclinEl. Differentiation experiment showed that CD11b expression on the surface of NB4 and U937-TR cells was upregulated after treatment with chidamide. Chidamide caused significant apoptosis of primary leukemia cells, but had a relatively weaker effect on normal hematopoietic cells. Colony forming assay revealed that the proliferation ability of TR mouse leukemia cells could be severely inhibited by chidamide. The in vivo study suggested that chidamide inhibited the proliferation of leukemia cell and significantly prolonged the survival time of leukemia mice when treated at the concentration of 12.5mg/kg and 25mg/kg. What’s more, during the period of drug treatment, body weight loss was barely observed which indicated that chidamide had low toxicity on recipient mice.Conclusions:As a novel HDAC inhibitor, chidamide can inhibit the proliferation of leukemia cells via inducing significant apoptosis, G0/G1 phase arrest and cell differentiation, which indicates that it has potential chemotherapeutic value in acute promyelocytic leukemia.