| Background and objective:Chronic lymphocytic leukemia (CLL) is a common middle-aged and old adult leukemia. Our previous works showed that TOSO was specifically overexpressed in patients with CLL and was an independent indicator for shorter survival.In addition, recent studies have demonstrated that TOSO may involve in the activation of NF-κB, MAPK pathway in other cells, which has been identified as the important abnormal pathways in CLL. But the detailed function of TOSO in CLL has not been well defined. Therefore, we will investigate the function of TOSO in the pathogenesis of CLL.Methods:1) After lentiviral transduction to B cell lymphoma’s cells(Granta-519and Z138), we investigate the effect of enhanced expression of TOSO on the proliferation,the cell cycle and apoptosis by flow cytometry. Western blotting (WB) was performed to detect the status of BCL-2, NF-κB and MAPK pathways.2) TOSO mRNA, Protein status and apoptosis were determined at48h after TOSO siRNA transfected into the primary cells of CLL.3) By immunoprecipitation and liquid chromatographycoupled with tandem mass spectrometry (IP/LCMS), we identified that TOSO might have direct interaction with some protein, which is the crucial activator of NF-κB, MAPK path way.This complex formation will be validated by coimmunoprecipitation (IP) experiments. And we will investigate the effect of TOSO on the expression and activation of this protein.4) After treatment with the IP validated protien inhabitor for48h in control (CON) and TOSO overexpression (TOSO) cells in culture, we detect the key proteins in the activation of NF-κB and MAPK pathways. We want to confirm whether TOSO active NF-κB and MAPK signaling pathway through the IP validated protein.5) Western blot analysis revealed the expression of Toll-like receptor-4(TLR4)-dependent signaling pathway proteins in CON and TOSO cells in culture with the addition of Lipopolysaccharide (LPS)(200ng/mL) for different times.6) CON group and TOSO group of B cell lymphoma’s cell line xenograft tumor model was established in NOD/SCID mouse and tumor growth was monitored.7) The expression of cyclinD1proteins in xenograft tumor tissues were detected by immunohistochemistry. The apoptosis of xenograft tumor tissues was detected by TUNEL analysis.Results:1) Enhanced expression of TOSO in B lymphoma’s cells promotes growth and survival, induces apoptosis decrease, but no effect in cell cycle. Cells overexpressing TOSO stimulate more BCL-2, p-p65,p-IKBa, p-ERK, p-p38, and p-Akt activation than CON cells.2) Silencing of TOSO in CLL primary cells is associated with apoptosis increase and decrease survival, inhibiting the expression of BCL-2, p-p65,p-IKBα, p-ERK, p-p38, and p-Akt.3) By mass spectrometry, we identified that TOSO might have direct interaction with SYK, which is the crucial activator of NF-κB, MAPK pathway. IP studies identity SYK(p-SYK) as a binging parter of TOSO in enhanced TOSO expression cells of B lymphoma and the primary cells of CLL,and abrogation of TOSO/SYK binding following treatment with SYK inhabitor R788in TOSO-expressing cells.In addition, TOSO is a regulator of SYK phosphorylation and activity in over expression TOSO B lymphoma’s cells and the primary cells of CLL4) SYK inhabitor R788can partly impair enhanced phosphorylation of p65, IKBa, ERK, p38, and Akt in overexpressing TOSO cells.5) Overexpression of TOSO enhance the LPS response (TLR4downstream signaling) in enhanced TOSO expression cells of B lymphoma.6) In mouse xenograft model of B lymphoma’s cells, TOSO group promotes tumor growth significantly from17days (P<0.05).7) Immunohistochemical study showed a positive expression of CyclinDl in TOSO and CON group, while TOSO group shown an decrease positive rate for TUNEL staining. Conclusion:1. TOSO decreases apoptosis and promotes the growth and survival of B lymphoma’s cells in vitro and vivo. Silencing of TOSO is associated with apoptosis increase in CLL primary cells.2. By mass spectrometry approach, we have identified SYK as a candidate TOSO-interacting protein. The TOSO-SYK complex formation is validated by coimmunoprecipitation experiments. Importantly, we show that the association of TOSO with SYK leads to enhanced SYK phosphorylation and subsequent activation of the downstream NF-κB, MAPK and AKT pathway in CLL.3. Overexpression of TOSO enhance the LPS response in B lymphoma cells. Objective:To explore the treatment options for younger than60years old adults with Ph/BCR-ABL positive acute lymphoblastic leukemia by comparing efficacy of different therapy.Methods:From January2001to June2012,42adult patients were enrolled in the study. All patients received combined with standard VDCP±L±imatinb(IM) as induction therapy and then received intensive consolidation with modified Hyper-CVAD/MA±IM. After achieved complete remission1(CRi),some patients who had appropriate donor received allogeneic hematopoietic stem cell transplantation (allo-HSCT),the other got sequential intensive consolidation±IM and followed by autologous HSCT(ASCT) in molecular CR(MCR) and received MM±VP±IM as maintenance therapy after ASCT. Overall survival (OS), disease free survival (DFS), relapse rate (RR) were analyzed. With the follow up to April30,2012, the clinical parameters.Results:The CR rate afterl cycle of induction chemotherapy was83.3%.39(92.9%) patients achieved CR. With a median follow-up of26.5(8-75) months, the median DFS and OS were (22±3.5) and (37±5.3) months, and cumulative RI were (43.7±9.7)%. All patients of CT Group relapsed. Two patients with IM therapy pre-and post ASCT maintain MCR for35and12months after ASCT. But the other three ASCT patients without IM died for relapse in one year. The transplant related mortality rate of allo-HSCT group was12.5%. The estimated OS at3year of allo-HSCT (n=16), ASCT (n=5) and CT (n=7) group were (66.7±12.2)%,(25±21.7)%and (16.7±15.2)%respectively (P=0.014). And the estimated DFS at3year of these groups were (56.3±12.4)%,(26.7±22.6)%and0%(P=0.002).Conclusion:IM combined with intensivechemotherapy significantly increased the CR rate and improved the quality of CR, which highly enhanced the feasibility of SCT. But it was poor for easy to relapse. Allo-SCT in CR1can decrease relapse and facilitates favorable OS and DFS of young adults with Ph+ALL. For those who without donor but achieved MCR, ASCT combined IM maybe a treatment option. |
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