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The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

FLT3-ITD mutations are seen in 20-30% of adult and 15% of pediatric acute myeloid leukemia (AML) patients and are associated with poor prognosis with higher rates of relapse and therapy resistance. FLT3 tyrosine kinase inhibitors (TKIs) have shown some success in treatment of FLT3-ITD AML, but responses are often not complete or sustained. Multiple potential mechanisms of drug resistance could be active in patients with FLT3-ITD AML including mutations in the FLT3 kinase, exogenous signaling from the bone marrow microenvironment, and altered epigenetic regulation. Mutations involving epigenetic modifiers such as DNMT3A, IDH1/2, TET2, ASXL1 and MLL1 are often co-expressed with the FLT3-ITD mutation. In addition, over-expression of EZH2, DOT1L and protein arginine methyl-transferase (PRMT) have also been reported. However, the role of different epigenetic mechanisms in regulating growth of FLT3-ITD AML cells and to modulate ability of TKIs to target FLT3-ITD AML cells has not been systematically studied. The objective of our study was to identify epigenetic targets in FLT3-ITD AML by screening a panel of epigenetic modifiers for their ability to inhibit growth of FLT3-ITD AML cells, and to enhance growth inhibition in combination with a FLT3 TKI. We used a carefully curated and well validated collection of epigenetic inhibitors (Structural Genomics consortium, Epigenetics Probes Collection, SGC Toronto; www.thesgc.org) that included 38 probes that target a spectrum of proteins involved in epigenetic regulation. We exposed MOLM-13 and MV4-11 human FLT3-ITD positive AML cells to each inhibitor by itself and in combination with the TKI Quizartinib/AC220 (500pM, IC20). Cell growth was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. The FLT3-ITD negative AML cell line OCI-AML3 (FLT3-WT) was studied as a control. The combined results of three experiments carried out using triplicate wells in each experiment were analyzed. Apoptosis was measured by flow cytometry using Annexin-V/FITC labeling and data analyzed by FlowJo V10. Statistical analysis was performed using GraphPad Prism 8.1.1 software. Of a total of 38 epigenetic inhibitors, less than a third resulted in reduction in cell proliferation after 72 hours of treatment (greater than 10% decrease, 13 for MOLM13, 9 for MV4-11 cell line). When used in combination with TKI (AC220), several epigenetic inhibitors resulted in significantly increased inhibition of proliferation of FLT3-ITD positive AML cells compared to either agent alone (21 for MOLM13, 10 for MV4-11 cell line) (p In conclusion, our studies using an unbiased approach have identified PRMT5 inhibition as a novel and selective approach to enhance targeting of FLT3-ITD AML cells in combination with FLT3 TKI. These results support our ongoing studies to evaluate the cellular and molecular mechanisms underlying these combinatorial effects, and to determine the translational therapeutic potential of this combination using primary patient samples and mouse models. Disclosures No relevant conflicts of interest to declare.

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained. Therefore, there is considerable interest in understanding mechanisms of resistance of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in resistance of FLT3-ITD+ AML stem cells to FLT3 TKI treatment. MV4-11 and Molm13 FLT3-ITD AML cells were transduced with GFP-LC3-RFP-LC3 ΔG constructs to measure autophagic flux. Autophagyhigh AML cells showed slower growth(p We tested the effect of Lys05 in vivo by treating NSG mice engrafted with primary FLT3-ITD AML cells with vehicle, AC220 (10mg/kg), Lys05 (20mg/kg) or combination for 3 weeks. Lys05 treatment reduced human CD45+ cell engraftment (8.7% vs 17%, p=0.03), and reduced CD45+CD33+ myeloid cells in BM and spleen (BM: 42% vs 81%, p We conclude that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML stem cells to TKI treatment both in vitro and in vivo. We demonstrate important roles for autophagy in regulating p53 levels and cell cycle in FL3-ITD AML cells. These studies support further development of strategies to target autophagy and related pathways to enhance efficacy of TKI in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

This file contains Tables S1â S7, including Table S1. CBF ratios in European, American, and Eastern Asian cohorts; Table S2. NPM1 mutation ratios in European and our cohorts; Table S3. FLT3-ITD mutation ratios in European and our cohorts; Table S4. FLT3-ITD mutation ratios in older patients from Chinese cohort against European and American cohorts; Table S5. CBF leukemia ratios in older patients from Japanese and Chinese cohorts against European and American cohorts; Table S6. NPM1 mutation ratios in older patients from Chinese cohort against European and American cohorts; and Table S7. Outcomes in European, American, and Eastern Asian cohorts. (PDF 568Â kb)

OBJECTIVE To explore the value of flow cytometric (FCM) analysis of cerebrospinal fluid (CSF) in the diagnosis of central nervous system involvement in adult patients with acute lymphoblastic leukemia (ALL) during follow-up. METHODS A total of 2871 CSF samples from 357 adult patients with newly diagnosed ALL between the year of 2009 and 2015 were analyzed retrospectively. These patients were divided into 3 groups according to CSF results, FCM+/conventional cytology (CC)+ group, FCM+/CC- group, and FCM-/CC- group, respectively. The overall survival (OS) of the three groups was analyzed. RESULTS Fifteen (4.2%) and 26 (7.3%) patients' CSF samples were FCM+/CC+ and FCM+/CC-, respectively. The remaining 316 (88.5%) patients' samples were FCM-/CC-. The 2-year OS for the FCM+/CC+, FCM+/CC-, and FCM-/CC- groups was 40.0%, 20.6%, and 64.2%, respectively (P

Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the deficiencies of conventional chemotherapy drugs and improve the efficacy. Many studies have revealed that the combination of histone deacetylase inhibitors (HDACi) and alkylating agents have synergistic effects. We reported a novel hybrid NL-101, in which the side chain of bendamustine was replaced with the hydroxamic acid of HDACi vorinostat (SAHA). NL-101 exhibited efficient anti-proliferative activity on myeloid leukemia cells especially Kasumi-1 and NB4 cells, accompanied by S phase arrest and caspase-3 dependent apoptosis. Importantly, it presented both the properties of HDAC inhibition and DNA damaging, as assessed by the acetylation of histone H3 and DNA double-strand breaks marker γ-H2AX. NL-101 also down-regulated the expression of anti-apoptotic protein Bcl-xL which was involved in the mitochondrial death pathway. Meanwhile, NL-101 induced apoptosis and DNA damage in primary cells from acute myeloid leukemia (AML) patients. NL-101 treatment could significantly prolong the survival time of t(8;21) leukemia mice with enhanced efficacy than bendamustine. These data demonstrate that NL-101 could be a potent and selective agent for leukemia treatment.

Hematopoietic recovery is considered to be associated with the number of multipotent hematopoietic stem cells in the bone marrow, as observed in functional assays involving stem cell transplantation. However, there is little evidence related to hematopoietic recovery in non-transplantation settings, which is accomplished by endogenous hematopoietic cells. A recent study suggested that progenitors are the main contributors during this steady-state hematopoiesis, which differs from exogenous transplantation. We hypothesized that endogenous progenitor support post-chemotherapy hematopoietic recovery. To investigate the potential impact of these progenitor cell percentage on hematopoietic recovery, we retrospectively analyzed the percentage of CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells (P cells) and hematopoietic recovery in 223 newly diagnosed acute myeloid leukemia patients during two courses of consolidation chemotherapy after complete remission. We found that a lower P cell percentage was significantly associated with prolonged neutropenia recovery time after the first and second courses of consolidation chemotherapy (p = 0.001; p = 0.045, respectively). We also observed similar results with regard to platelet recovery time after the first course of consolidation chemotherapy (p = 0.000). Univariate analysis showed that P cell percentage and consolidation chemotherapy regimens, and not gender, age, induction chemotherapy regimens, infection grade, WHO classification and NCCN risk category, were associated with neutrophil recovery after chemotherapy. Multivariate analysis demonstrated that P cell percentage is an independent factor affecting neutrophil recovery capacity for both the first and second courses (p = 0.008; p = 0.032, respectively). Our results indicate that CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells before each course of chemotherapy is independently associated with chemotherapy-related hematopoietic reconstitution capacity. These findings may help modify future chemotherapy regimens based on progenitor cell percentages.

Background The incidence of acute erythroid leukemia subtype (AEL) is rare, accounting for 5% of cases of acute myeloid leukemia (AML), and the outcome is dismal. However, in 2016 revision to the WHO classification, the subcategory of AEL has been removed. Myeloblasts are redefined as the percentage of total marrow cells, not non-erythroid cells. Therefore, the previously diagnosed AEL cases are currently diagnosed as AML or myelodyspalstic syndrome (MDS) according to new criteria. Methods We respectively reviewed cases of 97 de novo previously diagnosed AEL and all the patients were diagnosed as AML or MDS according to the new classification scheme, and then the clinical characteristics of these two subtypes were compared. Statistical analyses were performed by SPSS software version 18.0. Results The median age was 37 years-old, the two-thirds of previous AEL cases were diagnosed as MDS, and there was no obvious difference between two subtypes except for male/female ratio and age. Cytogenetic, rather than MDS/AML subtypes, can better represent the prognostic factor of previously diagnosed AEL patients. When the cytogenetic risk of patients belonged to MRC intermediate category and age were below 40 years-old in previous AEL cases, the patients who received induction chemotherapy without transplantation had a similar survival compared with the patients who underwent transplantation (3-year OS: 67.2% vs 68.5%). Conclusions Cytogenetic, rather than MDS/AML subtypes, can better represent the prognostic factor of previously diagnosed AEL patients. Transplantation was a better choice for those whose cytogenetic category was unfavorable. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3528-6) contains supplementary material, which is available to authorized users.

The ratio of different cytogenetic risk category in different age group. The ratio of different cytogenetic risk category (intermediate and unfavorable risk) in 60 age group. (DOCX 12 kb)

Raw data. The clinical characteristic of 97 previously diagnosed de novo adult acute erythroid leukemia patients. The clinical characteristic of 97 previously diagnosed de novo adult acute erythroid leukemia patients were listed, including MDS/AML subtype, MRC cytogenetic risk, survival data, gene mutation and so on. (DOC 239 kb)

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained in most patients. Therefore there is considerable interest in understanding mechanism contributing to resistance and preservation of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in FLT3-ITD+ AML cells, and its role in resistance to FLT3 TKI treatment. Expression of a FLT3-ITD construct in FLT3-ITD negative OCI-AML3 cells using lentiviral vectors resulted in increased autophagic flux compared with wild-type FLT3 or non-transduced controls. However, treatment of FLT3-ITD+ MV4-11 and Molm13 AML cells, or primary FLT3-ITD+ CD34+ cells, with the potent FLT3 TKI AC220 (Quizartinib) did not significantly alter autophagic flux. These results suggest that FLT3-ITD expression increases autophagy but in a kinase-independent manner. MV4-11 and Molm13 cells were transduced with the GFP-LC3-RFP-LC3 ΔG construct (Kaizuka T, et al,2016) to measure autophagic flux. Autophagyhigh AML cells showed lower growth rate compared with Autophagylow cells (0.65, p=0.025), and reduced sensitivity to inhibition by AC220 (0.49 vs 0.71, p=0.003). Treatment of MV4-11 and Molm13 cells with the potent autophagy inhibitor Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (MV4-11: 0.83 vs 0.54, p Induction of autophagy by starvation or mTOR inhibitor treatment in MOLM13 and MV4-11 cells was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression. Starvation-mediated autophagy in FLT3-ITD+ AML cells was significantly increased upon p53 knockdown, suggesting that p53 has an inhibitory effect on autophagy. Enhanced inhibition of FLT3-ITD+ AML cell growth by autophagy inhibition in combination with AC220 treatment was prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent. We conclude that FLT3-ITD expression enhances autophagic flux in FLT3-ITD+ AML cells in a kinase-independent manner, and that increased autophagy contributes to TKI resistance of FLT3-ITD+ AML cells. We show that autophagy inhibition can enhance sensitivity of FLT3-ITD+ AML stem cells to TKI treatment. Finally, we demonstrate important roles for autophagy in regulating p53 levels, and conversely for p53 in regulating autophagy and the effects of autophagy inhibition in FLT3-ITD+ AML cells. These studies support further development of strategies for targeting of autophagy and related pathways to enhance efficacy of TKI treatment in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in inducing remission and prolonging survival of CML patients, cures remain elusive because of persistence of primitive leukemia stem cells (LSC). We have reported that SIRT1, a NAD+ dependent deacetylase, is overexpressed in CML LSC and contributes to their maintenance and TKI resistance, related at least in part to increased p53 acetylation and transcriptional activity (Li et.al; Cancer Cell 2012). However, these studies using RNAi and pharmacological inhibitors were limited by possible off-target effects and limited duration of exposure. Therefore, to definitively delineate the role of SIRT1 in CML development, we developed a conditional SIRT1 knockout mouse model by crossing SIRT1 floxed mice (SIRT1fl/fl) with the Mx1-Cre strain to delete SIRT1 in hematopoietic stem cells (HSC), and crossed with transgenic BCR-ABL mice (BA Mx1-Cre SIRT1fl/fl). SIRT1 deletion in normal HSC did not inhibit normal hematopoiesis. In contrast, SIRT1 deletion in LSC profoundly inhibited the development of CML, and SIRT1-deleted CML mice did not develop evidence of morbidity and demonstrated significantly lower leukocytosis, neutrophilia and splenomegaly, and markedly improved survival, compared to controls (Fig-1). CML development in the SCL-tTA/BCR-ABL mouse model is associated with reduction in BM stem cells, and increase in stem/progenitor cell populations in the spleen. Splenic stem and progenitor populations were significantly decreased in SIRT1-deleted mice, whereas BM LTHSC were increased, indicating reversal of alterations associated with CML. SIRT1 deletion was associated with a significant decrease in LTHSC in G0 phase and increase in LTHSC in G1 phase of cell cycle. SIRT1-deleted LTHSCs generated significantly lower WBC counts, donor cell and donor LTHSC engraftment following transplantation into wild-type recipients compared to controls, indicating reduced in vivo repopulating ability. Treatment with the BCR-ABL TKI Nilotinib (50mg/kg/day) or vehicle for 4 weeks resulted in additional suppression of CML hematopoiesis in SIRT1-deleted mice. Gene expression analysis identified significant inhibition of mitochondria and oxidative phosphorylation related genes in SIRT1-deleted CML stem cells compared to controls. We performed extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF analyzer to evaluate mitochondrial respiration and glycolysis in c-Kit+ stem/progenitor cells from SIRT1-deleted and control CML and normal mice. CML LSC demonstrated significantly increased ATP-linked respiration, maximal mitochondrial respiration and mitochondrial reserve capacity compared to normal HSC. SIRT1-deletion resulted in significant reduction in basal and maximal mitochondrial respiration, and in mitochondrial reserve capacity, in CML LSC (Fig-2). In contrast, SIRT1-deletion did not result in significant change in mitochondrial respiration in normal HSC, suggesting that effects of SIRT1 deletion on mitochondrial respiration were specific to CML cells. The SIRT1 substrate PGC-1α is a master regulator of mitochondrial biogenesis and activity. Treatment of CML mice with the PGC-1α inhibitor SR-18292 significantly reduced basal, ATP-linked and maximal mitochondrial respiration. Consistent with results obtained with murine LSC, SIRT1 inhibition with TV39OH resulted in significant reduction in mitochondrial respiration in CML CD34+ cells compared to vehicle controls. Treatment of CML mice with Nilotinib did not significantly alter mitochondrial respiration in LSC. Similarly TKI treatment did not affect mitochondrial respiration in CML CD34+ cells compared to vehicle controls. In conclusion, our studies definitively demonstrate that SIRT1 is required for leukemia development, and reveal its critical role in increased mitochondrial respiration in CML LSC. Increased mitochondrial metabolism is kinase-independent, and is mediated by PGC-1a. SIRT1 inhibition results in enhanced inhibition of TKI-treated LSC. Our studies demonstrate an essential role for SIRT1 in altered mitochondrial metabolism in CML LSC, and support further exploration of SIRT1 pathway inhibition for LSC targeting. Disclosures No relevant conflicts of interest to declare.

Background As the important suppressor of P53, iASPP is found to be overexpressed in leukemia, and functions as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Methods Co-immunoprecipitation and immunofluorescence were perfomed to identified the interaction between iASPP and Sertad1 protein. Westernblot and Real-time quantitative PCR were used to determine the expression and activation of proteins. Cell proliferation assays, cell cycle and cell apoptosis were examined by flow cytometric analysis. Results iASPP combined with Sertad1 in leukemic cell lines and the interaction occurred in the cytoplasm near nuclear membrane. iASPP could interact with Sertad1 through its Cyclin-A, PHD-bromo, C terminal domain, except for S domain. Overexpression of iASPP in leukemic cells resulted in the increased cell proliferation and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs, the resistance to chemotherapeutic drug in iASPPhi leukemic cells was accompanied by Puma protein expression. Excess Sertad1 protein could tether iASPP protein in the cytoplasm, further reduced the binding between iASPP and P53 in the nucleus. Conclusions Sertad1 could antagonize iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells when iASPP was in the stage of overproduction. Electronic supplementary material The online version of this article (10.1186/s12885-017-3787-2) contains supplementary material, which is available to authorized users.

Objects: Investigate the clinical characteristics and factors of the impact on the survival of FLT-ITD mutation in the acute myeloid leukemia(AML) with intermediate chromosome karyoptye. Methods: We retrospectively collected and analyzed information regarding clinical characteristics and survival data from 133 patients with newly diagnosed AML with FLT3-ITD mutation and intermediate chromosome karyoptye from 2001-2015.The median follow-up time was 13.02 months. Results: 1. The median age was 37, median WBC was 50×109/L , the incidence between male and female was similar, the most common FAB subtype was M5(60.2%), the hepatosplenomegaly incidence was 8.4% . 2.The complete remission (CR) was 64.5% after first induction chemotherapy. CR could reached 74.4% or 57.1% when induction regimen included the medium dose cytarabine(100mg/m2 d1-4;1g/m2 q12h d5-7) or standard dose cytarabine(Ara-C)(100mg/m2d1-7) respectively(p=0.063). 3. Totally 131 patients received chemotherapy, and subsequently 22 patients received transplantation after chemotherapy. When the patients only received chemotherapy, 3-year OS was 39.9%, 3-year DFS was 44.8% and while in the patients who received transplantation, 3-year OS was 73.5%, 3-year DFS was 77.5%, the outcome was better (p 4. Univariable analysis showed that OS was related with age, hepatosplenomegaly, transplantation, WBC, the remission of induction chemotherapy. DFS was related with transplantation, courses for CR. Cox regression showed that transplantation and remission of induction chemotherapy were the independent prognosis factor. When the patient only received chemotherapy, FACS-MRD persisted negative was the independent prognosis factor. Conclusions: The FLT3-ITD de novo AML patients with intermediate chromosome karyoptye had unique characteristics. The patients who attained CR after induction chemotherapy and received transplantation would get better outcome, while in the patients who only received chemotherapy, FACS-MRD persisted negative implied better outcome. The induction regimen that contained medium Ara-C could reach higher CR , that may translated into better survival, therefore it may be the appropriate choice for FLT3-ITD AML patients. Disclosures No relevant conflicts of interest to declare.

Objects: Investigate the clinical significance of myeloblasts percentage in the de novo acute myeloid leukemia(AML) with erythroid hyperplasia from a single center of China Methods: We retrospectively collected information regarding clinical characteristics and survival data from de novo101 patients with newly diagnosed AML with erythroid hyperplasia (> 50% erythroid cells) from 2002-2016. The total patients were divided into two groups according to the myeloblasts percentage in total marrow cells. Cases with20% total myeloblast was defined as high-blast group. Median follow-up of patients was 10.03 months. Results:1.A total of 101 patients were diagnosed as de novo acute myeloid leukemia with erythorid hyperplasia according to WHO2008 criteria, 70 patients was classified as low-blast group, 31 patients was classified as high-blast group. Compared with high-blast group, in the low-blast group, the median age was older (39.8 vs 33.3, p=0.044), male was predominant (71.4% vs 48.4%, p=0.01). But complete remission (CR) after induction chemotherapy was lower in high-blast group(44.4% vs 57.4%, p=0.019).There were no difference in the white blood count(WBC), chromosome karyotype and incidence of various gene mutations. 2. The median survival time in the low-blast group and high-blast group was 39.19 months and 23.43 months respectively. 3-year overall survival(OS) was 53.1% and 43.4%(p=0.668), 3-year disease free survival(DFS) was 71.4% and 47.8% (p=0.297). 3. Univariable analysis showed that OS of 101 AML patients was correlated with prognosis subtype, remission of induction chemotherapy, transplant and relapse, and not related with myeloblast percentage exceed 20% of total morrow cells or not, WBC, age, gender and induction regimens. DFS of the all patients was correlated with the remission of induction chemotherapy and prognosis subtype. Multivariable analysis showed that OS was only correlated with remission of induction chemotherapy and transplant, DFS was only correlated with remission of induction chemotherapy. Conclusions: In the AML patients with erythroid hyperplasia with20% total myeloblast, but the survival was similar in both patients regardless of myeloblast percentage. The patients who attained CR after induction chemotherapy and received transplantation would get better outcome. Disclosures No relevant conflicts of interest to declare.

Background: Patients with AML who obtain first complete remission (CR) are subsequently treated with post-remission treatment, including chemotherapy or hematopoietic stem cell transplantation (HSCT). Suitable post-remission treatment in patients with intermediate-risk acute myeloid leukemia (AML) is remaining investigation. Objectives: To retrospectively investigatethe value of allo-HSCT in the patients with intermediate-risk AML. Patients and Methods: From August 2010 to January 2016, newly diagnosed adultpatients (15 to 55 years old) with AML were consecutivelyenrolled in a prospective trial (ChiCTR-TRC-10001202). Patients were assigned HAD containing conventional dose of cytarabine (homoharringtonine, cytarabine (Ara-c), and daunorubicin) induction therapy (Ara-C 100mg/m2, d1-7) or HAD containing intermediate dose Ara-c (Ara-C 100mg/m2, d1-4, 1.0g/m2, q12h, d5-7). All patients who achieved CR received either high dose Ara-c or intermediate dose Ara-c plus anthracyclines. Allo-HSCT was based on therapeutic responseand patients' intention. Patients received 6 courses of consolidation therapy if they did not receive HSCT. Minimal residual disease (MRD) was analyzed by multi-color flow cytometry after induction and consolidation therapy. Results: 181 enrolled patients were intermediate-risk AML (median age 36 years old). The median follow-up was 15.5 (0.5-67.9) months. One patient was lost to follow-up after induction, 180 patients were evaluated. 128 patients (71.1%) achieved CR after one induction cycle, 7 patients (3.9%) were early death, and 45 patients (25.0%) did not achieve CR. Forty patients received allo-HSCT, 33 of them were in CR1. The median time from diagnosis to transplantation was 6.5(3.9-16.0) months. Three years overall survival (OS) of transplantation group was higher than that of chemotherapy group (78.0% vs 48.0%, P = 0.000). Three years relapse-free survival (RFS) was 42.0% and 45.0% for patients in transplantation group and chemotherapy group, respectively (P = 0.196). However, taking transplantation time into account, the outcome was different. If the median transplantation time (6.5 months) was defined as cut-off value, there is no statistically difference of 3 years OS (77.0% vs 69.0%, P=0.182) and 3 years RFS (42.0% vs 53.0%, P=0.320) between transplantation and chemotherapy groups in the patients without relapse within 6.5 months. Twenty-one patients relapsed within 6.5 months, only one of them proceeded to allo-HSCT, and 1 year OS was 29.0%. For 45 patients who did not achieve CR after one induction cycle, 3 years OS was much better by allo-HSCT as compared with chemotherapy (85.0% vs22.0%, P = 0.008). In order to evaluate prognosis earlier, we analyzed MRD by flow cytometry after induction, first and second consolidation cycles in those achieving CR after one induction cycle. The patients with once or more MRD positive of three time points had significantly inferior 6 months RFS than those with continuous MRD negative (61.0% vs 92.0%,P = 0.005). Similar trend was observed on 1 year RFS (46.0% vs 76.0%, P = 0.005). Conclusion: According to the actual treatment, the OS of the patients with intermediate-risk AML was improved by allo-HSCT. When transplantation time was considered in the analysis, there was no difference between allo-HSCT and chemotherapy in survival. The patients with poor early response to chemotherapy (those did not achieve CR after one induction cycle) really benefited from allo-HSCT. The patients without continuous MRD negative after induction, first and second consolidation cycles trended to be early relapse, they might be another subgroup benefiting from allo-HSCT. Acknowledgments: This study was supported, in part, by State Key Program of National Natural Science of China (81430004), Foundation for Innovative Research Groups of the NaturalScience Foundation of China (81421002). Disclosures No relevant conflicts of interest to declare.

Background: Minimal residual disease (MRD) based on quantitativeRT-PCR has become an important prognostic factor for leukemia. There are few cohort based studies to investigate prognostic factors for t(8;21) AML, especially after MRD is integrated. Objectives: To retrospectively investigatethe prognostic factors in t(8;21) AML when MRD is integrated. Patients and Methods: From August 2010 to January 2016, newly diagnosed adultpatients (15 to 55 years old) with AML were consecutively enrolled in a prospective trial(ChiCTR-TRC-10001202). Patients were assigned standard dose HAD (homoharringtonine,cytosine arabinosine(Ara-c), and daunorubicin) induction therapy or HAD containing intermediate dose Ara-c. All patients who achieved CR received either high dose or intermediate dose Ara-c. Allogeneic SCT was based on c-Kit mutation and patients' intention. Patients received 6 courses of consolidation therapy if they did not receive transplantation. MRD based on quantitative RT-PCR was performed after induction therapy and each consolidation therapy. Results: 113 enrolled patients were t(8;21) AML (median age 33 years old).108 patients (95.6%) achieved CR. Six of them receivedallogeneicstem-cell transplantation in CR1.The median follow-up is 14.6(0.6-59.2) months. Three years relapse-free survival (RFS) and overall survival (OS) is 57.2% and 71.8%, respectively. Analyses of prognostic factors were performed. In univariate analysis, high WBC count (cut-off value at 25X109/L) (P=0.007), c-Kit mutation (P=0.017), and MRD positive during therapy (P=0.001)were adverse prognostic factors for RFS. But Age (cut-off value at 45 years old) (P=0.957), gender (P=0.418), induction regimen (P=0.055), and consolidation regimen (P=0.311) were not prognostic factors for RFS. MRDpositive during therapy (P=0.004) was the only adverse prognostic factors for OS. High WBCcount (P=0.824), c-Kit mutation (P=0.052), Age (P=0.596), gender (P=0.714), induction regimen (P=0.222), and consolidation regimen (P=0.299) were not prognostic factors for OS. In multivariate analysis, WBC count and MRD were independent prognostic factors for RFS. And MRD was the only prognostic factors for OS. MRD tests were performed in 83 patients after induction and during consolidation therapy. All of tests were positive in 37 (44.6%) patients. 46 (55.4%) patients achieved negative at least one of tests. The cumulative MRD negative was 3.6%, 15.7%, 34.9%, 39.8%, 47.0%, 50.6%, 55.4% after induction therapy, the first, the second, the third, the forth, the fifth, and the sixth consolidation therapy, respectively. Patients with negative MRD had better 3 years RFS (78.6% vs. 32.1%, P=0.001) and OS (85.3% vs. 55.2%, P=0.004) than patients with positive MRD. C-Kit mutation analysis was performed in 105 patients. 27(25.7%) patients had c-Kit mutation and 78(74.3%) patients had wild type c-Kit. 3 years RFS in patients with wild type c-Kit is superior to that in patients with c-Kit mutation (57.5% vs. 35.8%, P=0.017). There was no statistically difference between wild type and mutated c-Kit in 3 years OS (73.6% vs. 42.4%, P=0.052). MRD became negative during consolidation therapy in 66.1% (39/59) patients with wild type c-Kit, which is significantly higher than patients with mutated c-Kit (27.8%, 5/18, P=0.004). But there was no significant difference in RFS or OS between MRD negative and positive in c-Kit mutated patients (P=0.847 for RFS, P=0.11 for OS). To evaluate prognosis earlier, we analyzed MRD after induction therapy. MRD after induction therapy was performed in 76 patients. MRD decreased to ≤0.1%, >0.1% to≤1%, and >1% in 15 (19.7%), 45 (59.2%), and 31(40.8%) patients, respectively.Patients with MRD ≤1% after induction therapy had favorable 3 years RFS (75.4% vs. 29.7%, P=0.004) and OS (77.9% vs. 53.3%, P=0.013) compared with patients with MRD >1% after induction therapy.In multivariate analysis, in addition to c-Kit mutation and WBC count, MRD after induction therapy was still an independent prognostic factor for RFS. And MRD was the only prognostic factors for OS. Conclusion: MRD is an independentprognostic factor for OS and RFS for patients with t(8;21). Acknowledgments: This study was supported, in part, by State Key Program of National Natural Science of China (81430004), Foundation for Innovative Research Groups of the Natural Science Foundation of China (81421002) Disclosures No relevant conflicts of interest to declare.

Emerging studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). A lot of investigators have reported the identification of cell surface markers, such as CD123. Here, we report the identification of N -cadherin and Tie2 as LSCs markers. Inoculation of CD34 + CD38 − CD123 + N -cadherin + and CD34 + CD38 − CD123 + Tie2 + population can induce leukemia in NOD/SCID mice. The leukemic blast cells from the primary leukemic mice could also induce leukemia in the secondary transplantation. These findings suggested that N -cadherin and Tie2 were the important markers that can assist in leukemia development.

The cellular and molecular genetic aberrations of hematopoietic and lymphoid tissues are increasingly important in leukemia classification and are prognostically significant. Although some recurrent molecular cytogenetic abnormalities in AML have been extensively studied, others including t(7;11)(p15;p15) have not been well characterized. In this paper, seventeen AML patients with t(7;11)(p15;p15) were retrospectively reviewed for cell morphology, immuno-phenotype, cytogenetics as well as clinical features and prognosis. Among them, thirteen were female; nine were AML-M2. Six patients who were newly diagnosed were alive, one was lost for followed up and ten died. The median survival was 8 months. Taking together, AML with t(7;11)(p15;p15) is a rare and distinct disease. Most patients with this translocation are female at younger age and have special clinical and hematological characteristics such as M2-subtype of AML, easy to relapse and poor prognosis.

Introduction Chromosomal translocations involving nucleoporin NUP98 protein have often been described in acute myeloid leukemia (AML). Fusing NUP98 gene at chromosome 11 into class I homeoprotein HOXA9 gene at chromosome 7 through chromosome translocation was detected more than fifteen years ago. Translocation t(7; 11) (p15; p 15) is a rare but recurrent chromosome aberration in AML, which was first reported in 1982 as an additional translocation in a patient with Philadelphia chromosome-positive chronic myeloid leukemia (CML). Subsequent studies have indicated that this translocation was also found in de novo AML, CML and myelodysplastic syndrome (MDS).In this study, we retrospectively investigated the clinical and laboratory profile of this aberration. Methods: From August 1997 to July 2015, twenty-three (0.76%) of the approximately 3000 newly diagnosed patients with de novo AML at our hospital were found to have the chromosome translocation t(7;11) (p15;p15). The diagnosis was based on bone marrow cell morphology, cytochemistry, immunophenotyping and chromosomal analysis. Karyotypes of the 23 patients were analysed with R-banding technique. Transcripts of NUP98-HOXA9 were amplified from 9 patients with PCR. The PCR products were resolved on and extracted from 1% agarose gels and sequenced. Results Clinical and laboratory characteristics of patients with t(7;11)(p15;p15) Of the twenty-three patients with t(7;11) (p15;p15), fifteen were females and eight were males. The median age was 34 years, ranged from 18 to 67 years. According to FAB morphology criteria, eleven were M2 subtype, one of whom complicated with myelofibrosis; six were M5, five were M4, and one was M6. The blast count in bone marrow aspiration ranged from 27% to 86%. All the 23 cases expressed myeloid-associated antigen CD33, 21 cases expressed CD117 and CD13, HLA-DR and CD34 were expressed in 17 patients. None of the patients expressed lymphoid-associated antigens except two cases partially expressed CD9. Clonal chromosomal abnormalities of all the patients were t(7;11) (p15;p15). Additional trisomy 8 was found in only one patient. FLT3/ITD was positive in two of twenty-one patients. Outcome All the twenty-three patients received conventional induction chemotherapy including anthracyclines, cytarabine and homoharringtonine. The complete remission (CR) rate was 86.9% (20/23); the median follow up was 16 months; and the median disease-free survival and the median overall survival were 12 (0-45) months and 16 (2-50) months, respectively. Nine patients were still alive till last follow-up, one patient was lost for followed up and the other thirteen patients died. Among the nine survivals, four underwent sibling donor stem cell transplantation during CR1 period and two patients underwent Haplo-HSCT, unrelated donor allogeneic transplant for the seventh one, and the left two had shorter CR1 period because they were newly diagnosed. Among the thirteen dead patients, three did not achieve a complete remission and died during induction chemotherapy; one relapsed twice after underwent sibling donor stem cell transplantation three times and then died of acute graft-versus-host disease and gastrointestinal infection; one underwent sibling donor stem cell transplantation during CR1 period and then died of graft-versus-host disease and the other eight patients died after intensive chemotherapy including high-dose Ara-C, among whom, one with FLT3/ITD positive achieved CR1 for 8 months, and one died during treatment at the 29th month after relapsed twice at the 21th and the 27th months, respectively. Two types of fusion break points in NUP98-HOXA9 Four types of fusion transcripts have been previous reported as the consequence of alternative splicing.However, in our study, we only identified type I and type III fusion by sequencing. Type I included NUP98 ex.12 and HOXA9 ex.1b and type III included NUP98ex.11 and HOXA9 ex.1b. Conclusions Acute myeloid leukemia with t(7;11) (p15;p15) is a rare and distinct disease. Most patients with this translocation are female at younger age and have special clinical and hematological characteristics such as M2-subtype of AML, easy to relapse and poor prognosis. There are no target chemotherapy drugs to improve the prognosis so far. And whether stem cell transplantation is the therapeutic way for the disease need to be further investigated. Disclosures No relevant conflicts of interest to declare.

Introduction Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the flaws of conventional chemotherapy drugs and improve their efficacy. Histone deacetylase inhibitors (HDACi) and DNA damaging agents have showed synergistic effects in recent studies. In this study, we reported a novel hybrid NL-101 that combines chemo-active groups from suberoylanilide hydroxamic acid (SAHA) and bendamustine, the typical HDACi and alkylating agent respectively.The anticancer effect of NL-101 and its possible mechanisms were investigated in human leukemia cell lines and primary leukemia cells. Methods MTT assay was performed to determine the proliferation of Kasumi-1 and NB4 cells treated with NL-101. Cell cycle distribution and apoptosis rate were detected by flow cytometry. Western-blot analysis was used to analyze the level of acetylated H3 as well as apoptotic-related proteins including γ-H2AX, PARP, caspase-3, Bax, Bcl-2 and Bcl-xL. Bone marrow mononuclear cells of AML patients were isolated by density gradient centrifugation. Wright staining and Western blot were performed to determine the inducing apoptosis effect. Results NL-101 inhibited the proliferation of leukemia cell lines Kasumi-1 and NB4 cells with similar IC50 to that of SAHA. Cell cycle analysis indicated that NL-101 induced S phase arrest. As expected, apoptotic cell death was observed in response to NL-101 treatment. After treatment with 2 µmol/L NL-101 for 48 hours, the apoptosis rate of Kasumi-1 and NB4 cells were (60.19±12.01)% and (49.43±11.61)%, respectively. Western blot analysis showed that NL-101 exposure could induce the accumulation of acetylated Histone H3 and γ-H2AX as the biomarker of DNA double-strand breaks. Anti-apoptotic protein Bcl-xL involved in mitochondrial death pathway was also decreased. Moreover, NL-101 induced apoptosis with a low micromolar IC50 in various leukemia cell lines but not in nonmalignant cell line HEK293. The efficacy of NL-101 was also tested in human primary leukemia cells and all the treated samples exhibited apoptosis confirmed by the morphological examination and expression of apoptotic markers. Conclusions The novel SAHA-bendamustine hybrid NL-101 inhibited the proliferation and induced apoptotic cell death of leukemia cell lines and primary leukemia cells. It presented the properties of both HDAC inhibition and DNA damaging. Down-regulation of Bcl-xL was also involved in the apoptosis induction. These results indicated that NL-101 might be a potential compound for the treatment of leukemia. Disclosures Wang: Bristol Myers Squibb: Consultancy; Novartis: Consultancy.

Introduction: As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Sertad1 is highly expressed in carcinomas from pancreatic, lung and ovarian tissues, which considered Sertad1 as an oncoprotein. In this study, our findings revealed that Sertad1 could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus to interact with P53, and inhibited the function of iASPP eventually. Methods: Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the interaction between iASPP and Sertad1, the exact binding domains and the subcellular colocalization.The plasmids of iASPP and Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPPhi, K562-Sertad1hi, and K562-Douhi, respectively. The cell proliferation, cell cycle and apoptosis of above subclones were investigated by flow cytometry. Further, silence of the above two proteins was performed to confirm their functions. Immunoblotting analysis and immunofluorescence were performed to explore the possible mechanisms of difference between the biological functions of the above subclones. Results: Sertad1 expression level varied in leukemic cell lines and AML patients irrespectively of iASPP and P53. Interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells, both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP. Overexpression of iASPP in K562 cells (iASPPhi) could result in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time (Douhi) could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs. As figure showed, in K562-Douhi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. The immunoblotting analysis further supported the conclusions. The resistance of iASPP to chemotherapeutic drug was accompanied by Puma protein expression in a p53-independent manner. By knocking down the expersssion of iASPP and Sertad separately, we found that iASPP is dispensable for maintenance of anti-apoptotic function and Sertad1 is indispensable for cell cycle in leukemic cells. Conclusions: In normal situation, the protein iASPP and Sertad1 scatter in the nucleus and cytoplasm, mainly in the cytoplasm. As convinced by our study, iASPP was overexpressed in the leukemia cell lines and primary AML patients, it could function as oncogene through its binding with P53 protein in the nucleus, inhibit the function of P53. When iASPPhi cells were exposed to apoptosis stimuli, Puma protein could play an important role in this process, irrespective of the expression level of P53. But when iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, prevent it from inhibiting P53 function, suppress the leukemic cell growth and stimulate cell apoptosis by rescuing the P53 eventually. Our data provided a new insight to overcome iASPP protein, namely through its binding partners, when the similar proteins or drugs that can tether iASPP outside the nucleus such as Sertad1 are transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells. Figure 1 Figure 1. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.