ALBERT

Description: CD4+CD25+ regulatory T cells (Treg) play the critical role in maintenance of peripheral immune tolerance. However, the numbers of naturally occurring Treg (nTreg) that can be isolated from periphery are far too small to be clinically effective. The isolation and expansion of nTreg for treatment of autoimmune diseases encounter great difficulties. Whether autoantigen-specific Treg could be converted from CD4+CD25− T cells in patients with autoimmune diseases has not been reported. Here, we demonstrated that platelet glycoprotein (GP)–specific induced Treg (GP-iTreg) could be generated de novo from nonregulatory CD4+CD25−CD45RA+ cells in patients with idiopathic thrombocytopenic purpura and induced both antigen-specific and linked suppression. GP-iTreg mediated regulatory effects via modulating the T cell–stimulatory capacity of dendritic cells. By investigating the gene expression profile of iTreg-modulated dendritic cells, we provided a genome-wide assessment of the changes induced by antigen-specific iTreg and identified that the Toll-like receptor, Notch and transforming growth factor-β signaling pathways were related to the GP-specific tolerance, with the Toll-like receptor pathway being dominant. The findings in patients with idiopathic thrombocytopenic purpura will facilitate our understanding of the mechanisms of induction and maintenance of autoantigen-specific tolerance and highlight the considerable potential of antigen-specific iTreg for targeted immunotherapy in human auto-immune diseases.

Description: Idelalisib (Zydelig™), a first-in-class, selective, oral inhibitor of PI3Kδ, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation BTK inhibitor with improved selectivity compared to ibrutinib. We report here on the results of the combination of idelalisib and GS-4059 in lymphoma cell lines. Methods: Growth inhibition was assessed using CellTiter-Glo™ Assay (Promega) after 72-96 h incubation with idelalisib and GS-4059. Synergy for anti-proliferative effects was assessed using the Bliss Model of Independence (Meletiadis et al., Med Mycol, 2005), using MacSynergy II (Prichard et al., MacSynergyTM II, Version 1.0, 1993) or the Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Lysates were analyzed by Simple Western (Protein Simple) or Western blot. Ibrutinib resistance was established by continuous passaging of a clonal isolate of TMD8 in the presence of 10-20 nM ibrutinib. Resistance mutations were identified by whole exon sequencing (WES, GeneWiz). Results: GS-4059 potently inhibited growth (EC50

Description: Background Taking a step forward from the international prognostic index, attention is focused on the role of 18F-fluorodeoxyglucose positron-emission tomography-Computed Tomography scan (PET) as a tool for guidance in risk stratification in patients with diffuse large B-cell lymphoma (DLBCL). Here, we analyzed the predictive value of interim and post-treatment PET in patients with DLBCL. Methods In our retrospective study, we analyzed a homogeneous cohort of newly diagnosed DLBCL patients treated with R-CHOP21 who underwent PET scan at diagnosis, after three cycles of chemotherapy and at the end of treatment. For interim PET, images were interpreted by computing maximum standardized uptake value between PET performed at baseline and after three cycles of immuno-chemotherapy (¦¤SUVmax). The images of post-treatment PET were interpreted visually according to the International Harmonization Project criteria. The primary study endpoints were progression-free survival (PFS) and overall survival (OS). Results Thirty-eight patients with DLBCL with PET data were identified. The median follow-up was 28 months. At interim PET evaluation, optimal cutoff to predict progression was 66% for ¦¤SUVmax. ¦¤SUVmax analysis (≥66% vs〈 66%) identified patients with significantly different 2-year PFS (90.7% vs 38.9%; P =.000) and 2-year OS (95.5% vs 62%; P=.016) (Fig1). Post-treatment PET status (negative vs positive) was statistically significantly associated with 2-year PFS (91% vs 45.5%; P =.001) and 2-year OS (100% vs 72.7%; P =.012) (Fig2). Conclusions These data support the prognostic utility of PET in interim and post-treatment settings in patients with DLBCL treated with R-CHOP21. An interim PET scan after three cycles of treatment can effectively predict the outcome by using quantitative assessment of ¦¤SUVmax and allows clinicians to design a risk-adapted therapeutic strategy. A positive PET after the completion of therapy identifies a patient subset with an inferior PFS and OS. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction iASPP played an important role in leukemogenesis in our previous study. In order to clarify its mechanism, a yeast two-hybrid screen was performed to find the binding partner of iASPP. In this study, we reported FHL2 as a novel binding partner of iASPP. The biological functions of the communication between FHL2 and iASPP were detected, and its possible mechanisms were investigated in human leukemia cell lines. Methods A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP. Immunofluorescence, Co-IP and Western blot analysis were used to confirm the communication between FHL2 and iASPP. After FHL2 or iASPP was knocked down in K562 and Kasumi-1 cells by lentiviral, MTT assay and flow cytometry were performed to detect the proliferation, cell cycle distribution and apoptosis rate of leukemic cells, meanwhile Western blot analysis was used to analyze the level of cell cycle- and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when iASPP and FHL2 were overexpressed or FHL2 was knocked down. Results FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phases, and increase cell apoptosis. Western blot analysis showed that the level of p21 increased and anti-apoptotic protein Bcl-2 was reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax, furthermore, when FHL2 was knocked down at the same time, the transcriptional activity of p53 was rescued. Conclusions The interaction between FHL2 and iASPP in AML was observed for the first time. Cell proliferation reducing, cell cycle arresting at G0/G1 phases, and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown. Moreover, iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for AML treatment. Disclosures No relevant conflicts of interest to declare.

Description: Background Acute myeloid leukemia (AML) is a malignant hematologic disease with high incidence in the elderly peoples. The median age of onset is 65 years. There is no standard chemotherapy regimen for elderly AML, especially for AML patients older than 70 years old. Six to eight courses of low-dose or reduced-dose chemotherapy were commonly used in clinical treatment. However, most patients. However, most patients relapsed within six months after chemotherapy. How to prolong the survival of elderly patients with AML is a realistic problem that needs to be urgently solved. Patients and Methods From Jan 2017 to May 2018, six elderly patients with AML in our center include in the study. The median age was 74 (70-78) years. According to cytogenetics and molecular mutation, 1 patient were favorable risk with t (8, 21) and AML1-ETO, 3 patients were intermediate risk with karyotype abnormality, and 2 patients were unfavorable risk with complex karyotype and FLT3-ITD mutation. One patient received complete remission (CR) after IA induce scheme, and then, he received 4 courses of DA regimen for consolidation therapy. At last, he stopped chemotherapy because of severe atrial fibrillation and heart failure. Other 5 patients treated with 4-8 courses of decitabine (Dec) +CAG or HAG regimen. Minimal residual disease (MRD) of four patients were negative and two patients were positive before include in the study. Six patiens were given 10-day low dose Dec regimen treatment (5mg/m2/day×10 days) for every six weeks, until AML progress. Results For 2 MRD positive patients, after 10-day low dose Dec regimen treatment, one patient MRD turn to negative, one patient MRD remained positive, and died after 4 months. Till Jul 2018 (median observation time 10 months), 5/6 patients remained CR and survival with better quality of life. the most common treatment-emergent adverse events (TEAEs) were related to hematocytopenia. The most significant reduction of blood cells was hypoleucocytosis, and mainly in the first 2 courses of G-Dec treatment. Conclusion Preliminary research shows 10-day low dose Dec regimen treatment has Significant effect and mild side effect on the survival of elderly AML patients. The multicenter, randomized controlled clinical study will conduct to further verify its effectiveness and safety. Disclosures No relevant conflicts of interest to declare.

Description: Background Acute myeloid leukemia (AML) is a malignant hematologic disease with high incidence in the elderly peoples. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important method for the treatment of AML. However, elderly patients with AML are unable to receive myeloablative conditioning, and transplant related mortality (TAM) is significantly higher than that of young AML patients. How to reduce TAM in elderly AML patients, improve the efficacy of allo-HSCT, and further expand the applicable population of allo-HSCT is the focus of clinical research. Patients and methods According to the clinical trial (ChiCTR-IIR-16008182), elderly patients with AML used Decitabine (Dec) combined with FB3 as conditional regimen (20mg/m2 decitabine on day -11∽-7, 30mg/m2 Fludarabine on day -6∽-2, 130mg/m2 busulfan on day -4∽-2). The primary outcome of the trial was relapse after transplantation, and the secondary outcomes were chronic graft versus host disease (cGVHD) and the safety of treatment. Results From Mar 2016 to Jun 2018, 19 elderly patients with AML in our center include in the study. The median age was 62 (60-66) years. According to cytogenetics and molecular mutation, 8 patients were intermediate risk and 11 patients were unfavorable risk. HLA matched HSCT was performed in 7 patients, and HLA haploidentical HSCT in 12 patients. All of the patients achieved complete donor chimerism by day 28 after HSCT. Patients achieved ANC engraftment at 15 (10-19) days and PLT engraftment at 19 (12-26) days. Five patients relapsed after transplantation, and 4 of them died; one patient died of severe acute graft versus host disease (aGVHD) and pulmonary infection. The main adverse reactions during the conditioning treatment were dizziness, nausea, vomiting, diarrhea and oral ulcers. ALL symptoms were relieved after the symptomatic treatment. Three patients had a mild elevated transaminase and improved after liver-protecting treatment. Till now, the leukemia-free survival and overall survival were 73.68%. Conclusion Dec combined with FB3 as conditional regimen was well tolerated and can achieve ideal survival. The multicenter, randomized controlled clinical study will conduct to further verify its effectiveness and safety. Key Words: Decitabine, FB3, Elderly, Acute myeloid leukemia patient, Allogeneic hematopoietic stem cell transplantation Disclosures No relevant conflicts of interest to declare.

Description: Background CAR-T targeting CD19 has been a success in treating B-cell acute lymphoblastic leukemia (B-ALL). However, relapse rate is high and the long term survival in pateints is not satisfactory, which is partly due to the limited expansion and persistence of the conventionally-manufactured CAR-T cells. In addition, long manufacturing time and high cost of CAR-T product further limit the wider applications of CAR-T therapy. To solve these issues, we have developed a new manufacturing platform, FasT CAR-T, which shorten the manufacturing time to one day as compared to the conventional CAR-T manufacturing time of 9-14 days, which is critical for patients with rapidly progressing disease. More importantly, CD19-directed FasT CAR-T has been shown to have superior expansion capability, younger and less exhausted phenotype, and higher potency in eliminating B-ALL both in vitro and in vivo. Based on the preclinical study, we initiated a multi-center clinical study to determine the safety, feasibility and efficacy of CD19-FasT CAR-T in treating patients with CD19+ relapsed/refractory B-ALL. Methods CD19-directed CAR-T was manufactured using the FasT CAR-T platform. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis and T cells were separated. CD19-FasT CAR-T manufacturing were all successful. Conventional CAR-T (C-CAR-T) from healthy donor were also made in parallel for comparison in preclinical study. From Dec. 2018 to July 2019, 10 adult CD19+ R/R ALL patients were recruited and all patients received fludarabine and cyclophosphamide as pre-conditioning followed by a single CAR-T infusion 48-72 hours later. Doses used in this study were: 3 DL1 (5 x 104 CAR+ T/kg), 4 DL2 (1 x 105 CAR+ T/kg), and 3 DL3 (1.5 x 105 CAR+ T/kg). The endpoints of the study were clinical toxicity, feasibility, PK of CAR-T and efficacy. Results: In comparison to conventional CAR-T cells, CD19-FasT CAR-T cells had several key features (Table 1). 1) More robust expansion. Upon antigen stimulation, the FasT CAR-T proliferated 5-30 times stronger than that of C-CAR-T. 2) Higher percentage of CD62L+CD45RO- (Tscm) and CD62L+CD45+ (Tcm) population in FasT CAR-T. 3) Lower expression of PD-1+, LAG3+ and Tim3+ in FasT CAR-T. 4). More potent in eliminating Raji tumor in an in vivo xenograft mouse model. 5) More efficient migration to bone marrow which is likely due to the higher expression of CXCR4 on the FasT CAR-T cells. The trial was conducted during Dec. 2018 to July 2019. The pre-treatment bone marrow (BM) blasts were 〈 5% in 5 cases, 5%-50% in 3 cases, and 〉50% in 2 cases (Table 2). All 10 patients achieved complete remission (CR) 4 weeks after FasT CAR-T infusion, and 9 were with negative minimal residual disease (MRD-). CAR-T cells proliferation and persistence in peripheral blood (PB) were monitored by qPCR and flow cytometry. CAR-T cells peaked at Day 10 (range Day 8-13) after infusion. The median persistence period of CAR-T in PB was 56 days ((range 28-212 days) after infusion, and the longest persistence is 7 months and still being monitored at the last follow-up. The median peak of CAR copy number is 90,446/mg DNA (range 4,670-247,507/mg DNA) (Figure). The major adverse event was cytokine release syndrome (CRS) which was observed in 9 patients, including 1 patient with grade IV in DL3 group, 3 grade III, 4 grade II and 1 grade I. The clinical manifestation of CRS mainly included fever and hypotension. The median time to the development of CRS was 5 days (2-10 days), and the peak body temperature was at Day 7 (Day 5- 11) and fever lasted for an average of 5 days (3-8 days). Serum IL-6 level increased and peaked on Day 7 post-infusion, which coincided with fever but slightly preceded the CAR-T expansion peak. Three patients experienced CAR-related encephalopathy syndrome (CRES) after CAR-T infusion, in which 1 was grade III CRES. All patients who developed CRS or CRES recovered after intervention. Conclusion FasT CAR-T have superior expansion capacity with younger and less exhausted phenotype, and more potent cytotoxicity against B-ALL. This first-in-human clinical study in China showed CD19-directed FasT CAR-T therapy is highly effective in treating R/R B-ALL with manageable toxicity. The safety, efficacy and potential long-term clinical benefit of FasT CAR-T therapy will be further evaluated in large multi-center trial. (http://www.chictr.org.cn/listbycreater.aspx:ChiCTR1900023212) Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Idelalisib is a highly selective oral inhibitor of the phosphoinositide 3-kinase delta (PI3Kδ) that is hyperactive in many B-cell malignancies and is critical for the activation, proliferation, survival and trafficking of B lymphocytes. Idelalisib is approved in the US for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with relapsed follicular B-cell non-Hodgkin lymphoma and small lymphocytic lymphoma who have received at least two prior systemic therapies. Obinutuzumab (GA101) is a glycoengineered type II, CD20 antibody that induces a high level of direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcγRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLBCL. Previous work has shown the covalent BTK inhibitor ibrutinib can interfere with the immune effector function and ultimately in vivo efficacy of rituximab in preclinical models (Kohrt et al., Blood, 2014). As PI3K isoforms also play a role in immune effector cells and FcγR signaling we investigated the impact of PI3Kδ inhibition by the PI3Kδ selective inhibitor idelalisib on the immune effector function of obinutuzumab and rituximab. Experimental methods: The impact of idelalisib on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated in LDH release assays using WIL2-S, SU-DHL4 and Z138 target cells at plasma protein-binding adjusted clinically relevant concentrations mimicking exposure in patients. As a surrogate for NK cell activation CD16 levels and up-regulation of the degranulation marker CD107a were assessed by FACS. The impact on monocyte-derived macrophage mediated ADCP of WIL2-S cells was measured in a flow cytometry-based phagocytosis assay. Finally, depletion of CD19 positive B cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. Results: In ADCC assays, no impact of idelalisib on ADCC at saturating concentration of obinutuzumab or rituximab (〉1ug/ml) can be detected in LDH release assays with tumor cells targets (N=9 donors for WIL2-S, N〉3 donors for SU-DHL-4 and Z138). Idelalisib did not alter obinutuzumab or rituximab ability to kill tumor cells by ADCC at low E:T ratio. Little to no increase of obinutuzumab or rituximab EC50 for LDH release, CD16 down regulation, or degranulation of NK cells could be detected depending on donor effector cells. ADCP assays were conducted with M2c polarized macrophages using WIL2-S as targets. Less than 30% inhibition of ADCP was observed in this assay at idelalisib concentration at protein binding-adjusted clinical Cmax. At idelalisib Cmax (4200 nM) the EC50 of obinutuzumab-mediated B cell depletion in healthy human whole blood was increased 3 to 5 times, whereas at Cmin (760 nM) idelalisib did not significantly influence obinutuzumab EC50 or maximal B cell depletion. Conclusions: PI3Kδ inhibition by idelalisib has minimal impact on the immune effector function of obinutuzumab (GA101) and rituximab as measured in NK cell-mediated ADCC, macrophage-mediated ADCP and whole blood B-cell depletion. Disclosures Herter: Roche: Employment. Palazzo:Gilead Sciences: Employment. Bacac:Roche: Employment. Grosmaire:Gilead Sciences: Employment. Frey:Gilead Sciences: Employment. Pflanz:Gilead Sciences: Employment. Liu:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Queva:Gilead Sciences: Employment.

Description: Background: Idelalisib (Zydelig®), a potent and selective PI3Kδ inhibitor, was recently approved for the treatment of relapsed CLL, SLL and FL. PI3Kδ is critical for malignant B-cell proliferation, survival and homing, and inhibition results in a rapid decrease in lymphadenopathy and clinical response (Yang et al., Clin Can Res 2015). Although idelalisib is very potent in mitigating disease, most patients experience incomplete response and ultimately progress. To understand the mechanism of resistance, we screened ABC-DLBCL cell lines for sensitivity to idelalisib and selected TMD8, an ABC-DLBCL cell line, for the generation of idelalisib resistant cells. We report on the mechanisms of resistance and on downstream biomarkers associated with sensitivity to idelalisib in lymphoma cell lines. Methods: Growth inhibition to idelalisib or other inhibitors was assessed using CellTiterGlo viability assay (Promega) at 96 h. Idelalisib resistant line (TMD8R) was generated by continuous exposure to1μM idelalisib (~2x Cmax corrected for protein binding). A DMSO passage matched line was generated as control (TMD8S). Clonal isolates from pools were generated through 2 rounds of limiting dilution. Cell lines were analyzed by whole exome sequencing (WES, GeneWiz), RNASeq (Expression Analysis) and phosphoproteomics (MultiPathway PTMScan Direct, Cell Signaling Technologies). Protein expression was measured using Simple Western (Protein Simple) and SDS/PAGE Western blot. Results: TMD8 were sensitive to idelalisib and the pan PI3K inhibitor (GDC-0941) with an EC50 of 42 and 27nM, respectively, but not to PI3Kα (BYL-719) or PI3Kβ (AZD-6482) inhibitors, indicating that cell viability is mostly driven by PI3Kδ. TMD8 cells with acquired resistance to idelalisib (TMD8R) showed a loss of sensitivity to idelalisib, with a growth inhibition of 19% vs. 92% at 1μM as compared to TMD8S. No mutation in PIK3CD or other PI3K pathway members, including PTEN, was found in TMD8R clones by WES. In 8/8 TMD8R clones a dramatic reduction of PTEN protein expression was observed (9-fold) despite normal expression of PTEN mRNA by RNAseq (Figure 1A). Additionally 8/8 TMD8R clones showed a modest up-regulation of PIK3CG mRNA (2-fold) and protein (3-5 fold, Figure 1B). PIK3CD remained the most prevalent PI3K isoform expressed in all TMD8R clones (Figure 1C). TMD8R were cross-resistant to the dual PI3Kδ/γ inhibitor duvelisib (EC50 〉 4 uM for TMD8R vs. EC50 = 58 nM for TMD8S). RNAseq analysis of idelalisib sensitive and resistant ABC-DLBCL cell lines showed that idelalisib treatment led to c-Myc mRNA down regulation in sensitive cell lines (TMD8 and Ri-1) but not in the resistant cell lines (U2932 and SU-DHL-8). In addition, expression of c-Myc target genes was unchanged in the TMDR. Analysis of phosphoproteins showed PI3K and MAPK pathway up-regulation in TMDR, compared to TMD8S, with increased expression of p-Akt T308, p-Akt S473, p-PDK1 S241, p-GSK3β S9 and p-ERK T202/Y204 (11-, 6-, 2-, 1- and 8-fold, respectively), providing a potential mechanism for the loss of c-Myc down regulation in resistant cells (Wen-Bin Tsai et al., Cancer Res, 2012). Other B-cell receptor signaling proteins (p-BTK Y223, p-SYK Y525/526, and p-STAT-5 Y694) were not detected or unchanged. TMD8R cells were cross resistant to the BTK inhibitors ibrutinib and ONO/GS-4059. Akt (MK-2206) or PDK1 (GSK233440) inhibitors were less potent in TMD8R vs. TMD8S cells, yet in combination with idelalisib at 1μM, their potency was restored to the level observed in TMD8S, indicating that resistant cells retain some dependency on the PI3Kδ. Conclusions: Resistance to idelalisib in the ABC-DLBCL cell line TMD8 was not acquired through de novo mutations. Rather loss of PTEN protein expression, modest up regulation of PI3Kγ, activation of the PI3K and MAPK pathway and loss of c-Myc down regulation by idelalisib were identified as potential mechanism of resistance. Treatment with agents targeting the PI3K pathway in combination with idelalisib may overcome resistance. Figure 1. Profiling of TMD8R Cells Reveal Increased PI3Kγ and PTEN loss. (A) Decreased expression of PTEN (9 fold) in TMD8R clones as compared to TMD8S (B) Overexpression of PI3Kγ (3-5 fold) in TMD8R clones as compared to TMD8S (C) PI3Kδ isoform is the highest expressed isoform in both TMD8S and TMD8R clones. Figure 1. Profiling of TMD8R Cells Reveal Increased PI3Kγ and PTEN loss. (A) Decreased expression of PTEN (9 fold) in TMD8R clones as compared to TMD8S (B) Overexpression of PI3Kγ (3-5 fold) in TMD8R clones as compared to TMD8S (C) PI3Kδ isoform is the highest expressed isoform in both TMD8S and TMD8R clones. Disclosures Meadows: Gilead Sciences: Employment, Other: Share holder. Rick:Gilead Sciences: Employment, Other: stock holder. Anella:Gilead Sciences: Employment, Other: Stock holder. Liu:Gilead Sciences: Employment, Other: stock holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Stock holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.

Description: Backgrounds Leukemic stem cells (LSCs) have emerged as one of reasons for relapse to chemotherapy. To eradicate these quiescent LSCs is of great importance. Administration of granulocyte colony stimulating factor (G-CSF) with low-dose chemotherapy, as a priming strategy, has shown satisfactory effect in acute myelocytic leukemia treatment, especially in elder patients. It is important to investigate the specific signaling pathways and explore useful biomarker to monitor clinical response to therapy. MiR-146a and its downstream putative targets CXCR4 and Smad4 may sustain quiescent LSCs in bone marrow niches. The objective of this study is to further explore the possibility of miR-146a as a marker for sensitivity detection to G-CSF priming chemotherapy. Patients and Methods MiR-146a over-expression (OE), knock-down (KD) and empty virus (EV) HL-60 cells were constructedin vitro. Leukemia cells were pre-treated with G-CSF for 48h or not. MiR-146a, p50, p65, CXCR4 and Smad4 expression levels were detected. Human stromal cell line HS-5 and HL-60 cells were co-cultured to simulate interaction between bone marrow microenvironment and leukemia cells. Chemotaxis, cell viability, cell cycle and apoptosis were estimated individually. Relationship between miR-146a expression and prognostic factors were analyzed in 46 new diagnosis AML patients, including overall survival (OS) rates, relapsed-free survival (RFS) rates and complete remission (CR) rates. Result MiR-146a-OE cells showed lower CXCR4 and Smad4 levels than miR-146a-EV cells (p=0.011 and 0.01), while miR-146a-KD cells showed increased CXCR4 and Smad4 expression (p=0.0002 and 0.001), both in mRNA and total protein levels. Higher miR-146a expression was observed in G-CSF pre-treated group. p50 and p65 mRNA and total protein levels were both elevated in response to G-CSF treatment (p