ALBERT

Description: Abstract 45 Background Homoharringtonine-based induction regimens have been widely used in China for patients with acute myeloid leukemia (AML), which have shown to improve the rate of complete remission (CR) and long-term survival. We aimed to further evaluate its efficacy and safety in treatment of de novo AML. Methods This phase 3 study was done in 17 institutions in China. Patients between the age of 14 and 59 with untreated AML were randomly assigned to receive HAA (homoharringtonine 2 mg/m2/day, days 1–7; cytarabine 100 mg/m2/day, days 1–7, aclarubicin 20 mg/day, days 1–7), HAD (homoharringtonine 2 mg/m2/day, days 1–7; cytarabine 100 mg/m2/day, days 1–7; daunorubicin 40 mg/m2/day, days 1–3) or DA (daunorubicin 40–45 mg/m2/day, days 1–3; cytarabine 100 mg/m2/day, days 1–7) regimen as induction therapy. Patients who achieved partial remission or had a decrease of blast ¡Ý60% could receive a same second induction course. All patients who had a complete remission were offered the same consolidation chemotherapy according to the cytogenetic-risk. The primary endpoints were CR and event-free survival (EFS). The trial is registered in Chinese Clinical Trial Register, number ChiCTR-TRC-06000054. Results 620 patients were randomly assigned to receive HAA (n=207), HAD (n=206) and DA (n=207) regimens. HAA or HAD regimen, as compared with DA regimen, resulted in a higher rate of CR in the first course of induction therapy (67.5% vs. 54.0%, P=0.005; 64.9% vs. 54.0%, P=0.026, respectively). The overall CR rate remained significantly higher in the HAA arm as compared with DA arm (75.0% vs. 61.9%, P=0.005). HAA or HAD regimen has similar rates of adverse events as compared with DA regimen, but was associated with significantly increased risk of induction death (5.8% vs. 1.0%, P=0.007; 6.6% vs. 1.0%, P=0.003, respectively). The EFS was greatly improved in the HAA arm (3-year EFS 35.4±3.5% vs.23.1±3.1%, P=0.002), while not significantly in the HAD arm (3-year EFS 32.7±3.5% vs.23.1±3.1%, P=0.078) as compared with the DA arm. Overall survival (OS) and relapse-free survival (RFS) did not differ significantly in the HAA or HAD arm as compared with DA arm, but an OS and RFS advantage of the HAA arm over the DA arm was observed in patients with favorable or intermediate cytogenetic profile (OS: P=0.014; RFS: P=0.022, respectively). Patients in the HAD arm with NPM1 but not FLT3ITD mutations, as compared with the patients in the DA arm, had an improved EFS (P=0.038). In intermediate cytogenetic profile, patients with mutant CEBPA had prolonged RFS in the HAA arm as compared with the DA arm (P=0.045). Conclusions Homoharringtonine-based induction regimens are associated with a higher rate of CR and improved survival as compared with DA regimen in AML. The toxicity is mild with the exception of a higher rate of induction death. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points NrasG12D/+ induces proliferation and increases self-renewal and myeloid differentiation bias in HSCs. ERK1/2 is constitutively hyperactivated in NrasG12D/+ HSCs and downregulation of the MEK/ERK signaling attenuates NrasG12D/+ HSC phenotypes.

Description: Key Points Role for LIMK1 in GPIb-IX–dependent cPLA2 activation, TXA2 synthesis, and platelet activation independent of its role in actin polymerization. LIMK1 is important in arterial thrombosis in vivo but appears to be dispensable for hemostasis, suggesting a new antithrombotic target.

Description: Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1-/- platelets. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Deletion of Fanca or Fancd2 dysregulates the activity and function of regulatory T cells. The loss of FA proteins alters the expression of Foxp3 target genes.

Description: Background Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), a soluble N-terminal fragment of GPIbα, has important implications for thrombosis and hemostasis. Stimulations of platelet with either physiological or chemical compounds result in GPIbα ectodomain shedding in vitro and in vivo. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen, thrombin, and thromboxane A2 analog U46619. In this paper, we plan to clarify the relationship between physiological stimulation-induced ROS generation and GPIbα shedding and the regulatory mechanism of ADAM17-mediated GPIbα shedding. Methods Washed platelets (3×108/ml) were pre-incubated with or without N-acetylcysteine (NAC) (10 mM), dithiothreitol (DTT) (3 mM), or vehicle control (DMSO) at RT for 15 minutes, and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 °C (or at RT for dibucaine) for different time. Western blot and flow cytometry was used to evaluate the GPIbα ectodomain shedding. ROS levels in platelets were examined using ROS assay kit according to the manufacture’s instruction. Results and conclusions Collagen, thrombin, and calcium ionophore A23187 induced ROS generation, and simultaneously incurred GPIbα ectodomain shedding. ROS scavengers NAC and DTT abolished not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induced both ROS production and GPIbα shedding, which were also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal (MDL), obviously reduced dibucaine-induced ROS production, and inhibited ROS generations induced by A23187 and thrombin. These data indicate that ROS, regulated by calpain, plays a key role in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug. Disclosures: No relevant conflicts of interest to declare.

Description: Adult hematopoietic stem cells (HSCs) primarily reside in the hypoxic bone marrow microenvironment, and preferentially utilize anaerobic glycolysis to obtain energy. Cited2 is a cytokine-inducible gene, which plays various roles during mouse development. Our previous studies showed that deletion of Cited2 in adult mouse results in loss of HSC quiescence, increased apoptosis, and impaired HSC reconstitution capacity (Blood 2012, 119:2789-2798). In this study, we conditionally deleted Cited2 in Cited2fl/fl;Mx1-Cre mice and investigated the role of Cited2 in the metabolic regulation of HSCs. First, we examined mitochondrial alterations in Cited2 knockout (KO) long-term (LT-) and short-term (ST-) HSCs defined as “Flt3-CD34- LSK” and “Flt3-CD34+ LSK”, respectively. Staining with MitoTracker Green revealed that deletion of Cited2 resulted in a significant increase in mitochondrial mass in both LT- and ST-HSCs but not in the whole bone marrow cells. To explore the morphological changes of mitochondria in Cited2 KO HSCs, we sorted Flt3-LSK cells (containing LT- and ST- HSCs) and performed electron microscopy ultrastructural analysis. The mitochondria in wild type (WT) HSCs were mostly small, round or oval, and dark (Figure 1). However, Cited2 KO HSCs displayed markedly elongated and brighter mitochondria, similar to those observed in aged WT HSCs (20–24 months old mice) by others. The frequency of Cited2 KO LT-HSCs with high mitochondrial membrane potential was significantly increased (8.5% in WT versus 15.1% in KO). Furthermore, the reactive oxygen species (ROS) levels in Cited2 KO HSCs were significantly higher than those in WT controls. To further understand the metabolic changes in Cited2 KO HSCs, we measured glucose uptake using fluorescent indicator 2-NBDG. Glucose uptake was unchanged in the Cited2 KO LT- and ST- HSCs. Also, intracellular ATP content was maintained at the normal levels in Cited2 KO LT-HSCs, although slightly increased in ST-HSCs compared with WT controls. To assess the utilization of glycolysis in Cited2 KO HSCs, glycolytic flux was determined by glucose-derived 13C-lactate production using Gas Chromatography–Mass Spectrometry (GC-MS). We found that the rate of 13C-lactate production was significantly lower in both LT- and ST-HSCs lacking Cited2 than in WT controls. To further confirm this finding, we treated HSCs with antimycin A (AMA), a specific inhibitor of mitochondrial electron transport chain. We found that Cited2 KO HSCs displayed increased NADH after AMA treatment, compared with the WT control, indicating that mitochondrial respiration was increased in KO HSCs and produced more NADH. At the molecular level, deletion of Cited2 significantly reduced the expression of metabolism related genes in HSCs, such as lactate dehydrogenase (LDH) B and LDHD, pyruvate dehydrogenase kinase (Pdk) 2 and Pdk4, PYGL (phosphorylase, glycogen, liver), and GPX1 (glutathione peroxidase 1). Notably, Pdk2 and Pdk4 were recently shown to be critical controllers of glycolysis and checkpoint for cell cycle in HSCs. Consistent with reduced expression of Pdk, the phosphorylation of PDH-E1α was significantly decreased in Cited2 KO HSCs. Akt, mTOR, and FoxOs are known regulators of mitochondrial functions in HSCs. We found that Akt-mTOR signaling activity was increased in Cited2 KO HSCs, as indicated by increased phosphorylation of Akt and S6 ribosomal protein. However, in vitro treatment of LT-HSCs with mTORC1 inhibitor rapamycin did not resume decreased expression of LDHB, LDHD, Pdk2, and Pdk4, suggesting that elevated mTORC1 activity may not be the major contributor to the downregulation of glycolysis related genes. Meanwhile, we also found that in Cited2 KO LT-HSCs, phosphorylation of FoxO1 and FoxO3 was increased, both of which are known regulators of Pdk4 expression. Interestingly, in vitro treatment of LT-HSCs with PI3/Akt inhibitor LY294002, partially rescued the expression of Pdk4. Together, these findings suggest that the downregulation of Pdk4 in Cited2 KO HSCs is likely mediated by the inactivation of FoxOs caused by the elevated Akt activity. In summary, these results show that loss of Cited2 attenuates HSCs' glycolytic metabolism while simultaneously enhancing their overall mitochondrial oxidative phosphorylation, thus suggesting a critical role of Cited2 in the maintenance of adult HSC glycolytic metabolism likely through regulating LDH, Pdk, and Akt activity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4725 Chemotherapy is widely used in treatment of myeloid leukemia, the efficancy of which, however, is often hampered by the development of intrinsic and acquired multidrug resistance(MDR), the exact mechanism of which is still unclear. Overexpression and deregulated activation of protein tyrosine kinase(PTK) are frequently observed in several types of hematologic malignancies, the abnormally signal cascade transducted by which has been demonstrated to play an important role in antiapoptosis, differentiation block, enhancing autonomous proliferation, and also in inducing drug resistance. EphB4 is also a protein tyrosine kinase, a member of the largest family of receptor tyrosine kinase, which has been found with abnormally upregulated expression or activity in many types of cancer especially solid tumor types, such as mammary adenocarcinoma, colon carcinoma, ovarian cancer and prostatic carcinoma. Here the aim of our study was to examine the expression and biological role of EphB4 in drug resistance of myeloid leukemia. Using RT-PCR assay and western blot, we tested the mRNA level in 35 myeloid leukemia patients including 4 cases of acute myeloid leukemia(AML) with primary multiple drug-resistance(MDR), 3 cases of AML which have relapsed, 5 cases of newly diagnosed AML, 5 cases of AML which got (complete remission) CR at the first chemotherapy treatment, 9 cases of chronic myeloid leukemia in chronic phase(CML-CP), and 9 cases of CML in blast crisis(CML-BC). The results showed the EphB4 mRNA level was significantly upregulated in AML bearing relapse(EphB4/β-actin ratio:0.962±0.114) or MDR(EphB4/β-actin ratio: 0.993±0.047) and also in CML-BC(EphB4/β-actin ratio: 1.001±0.060) compared with newly diagnosed AML(EphB4/β-actin ratio: 0.332±0.014), AML with CR(EphB4/β-actin ratio:0.401±0.015) and CML-CP(EphB4/β-actin ratio: 0.432±0.020). Subsequently, we examined both the transcriptional and translational level of EphB4 in several myeloid leukemia cell lines including K562, HL60,U937,KG1α and an adriamycin-resistant cell line—HL60/ADM, and drug-resistant capacity of the five cell lines was also tested by CCK-8 assay. Finally EphB4 protein expression is found to be upregulated at both transcriptional and translational level in K562, KG1αand HL60/ADM, which showed stronger capacity of resistance to gradient concentrations of adriamycin(IC50 of K562:0.451±0.037ug/ml,KG1α:0.217±0.017ug/ml, HL60/ADM: 2.663±0.102ug/ml) compared with that of HL60 and U937(IC50 of HL60:0.040±0.001ug/ml, U937:0.040±0.005ug/ml) in which little or no expression of EphB4 mRNA or protein was observed. And the most noteworthy is that HL60/ADM, which shows the strongest drug resistant capability. also bears the most amount of EphB4 at both transcriptional level (EphB4 /β-actin ratio: 1.002±0.017), and translational level (EphB4 /β-actin ratio: 0.975±0.051). These data supports a role for EphB4 in inducing drug resistance and raise the possibility that therapeutic intervention to EphB4 expression or signaling might inhibit or even reverse drug resistande in meyloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: The benefit of radiotherapy (RT) following chemotherapy in limited-stage DLBCL remains controversial. Before the Rituximab era, 4 randomized trials have been reported with conflicting results (ECOG 1484 and SWOG 8736, GELA 93-1 and 93-4 studies). More recently, the German Unfolder study prematurely closed the R-CHOP without RT arm in bulky limited-stage DLBCL due to an excess of relapse. In 2005, we conducted a randomized trial in patients with non-bulky (defined by a tumor size 50% but a persistent positive FDG-PET) after C4, 2 additional cycles of R-CHOP followed by RT (even if not initially allocated) were recommended. The primary objective was EFS at one year after the last randomization, and secondary objectives were the impact of interim FDG-PET on EFS and the toxicity of RT. From May 2005 to December 2013, 313 patients were randomized and 301 patients are currently evaluable. Median age was 56 yr (20-75). There were 181 males and 120 females: 106 patients (35%) were older than 60 yr. Most patients had normal LDH (82%), PS=0 (80%), and no B symptoms (96% of cases). Modified IPI score was as follows: IPI =0 (n=170), IPI=1 (n=113), IPI=2 (n=16), IPI=3 (n=2). Main tumor sites were cervical (n=159), Waldeyer’s ring and sinus (n=36), inguinal (n=29), axillary (n=25), mediastinum (n=21). Extra-nodal sites were observed in 121 patients (40%). One hundred and fifty patients were randomized in the R-CHOP arm and 151 in the R-CHOP + RT arm. After 4 cycles, 253 patients (84%) were in CR and 43 in PR (14%). Three patients had stable disease. Thirty-four patients (79%) out of the 43 partial responders received 2 additional cycles of R-CHOP followed by RT (including 12 patients not initially allocated to RT arm). At the end of treatment, CR and PR rate were 94% and 3%, respectively. Seven (4%) out of the 151 patients randomized in the RT arm declined radiation. With a median follow-up of 51 months (2-110), there were 20 relapses: 12 in the R-CHOP arm and 8 in the R-CHOP+RT arm (p=ns). Sixteen patients died. Causes of death were as follows: relapses (n=9), toxic (n=1), secondary malignancies (n=3), unknown (n=3). Median time of relapse was 21 months (2-110 months). EFS and OS are not statistically different between the two arms. In an intent to treat analysis, 5y-EFS is 87% n the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.55), p=0.13, and 5yr-OS is 90% in the R-CHOP arm versus 95% in the R-CHOP + RT arm (HR=0.60), p=0.32. For patients in complete response after the 4 cycles of R-CHOP (84% of the patients), 5yr-EFS is 89% in the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.59), p=0.24. In this prospective study, the results demonstrate that in non-bulky limited-stage DLBCL, R-CHOP alone (4 to 6 cycles) induces very high CR rate with a very good overall survival and a very low relapse rate. With the current follow-up, the addition of radiotherapy is not significantly superior to R-CHOP alone and should be reserved to the minority of patients who do not reach CR after R-CHOP. Disclosures Gyan: Roche: Research Funding.