ALBERT

Description: Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome caused by excessive immune activation leading to a life-threatening pro-inflammatory cytokine storm. HLH is not an entity of its own, but a clinical syndrome triggered by various conditions like infections, malignancy or autoimmune disorders. HLH pathogenesis is complex and still not fully understood. Contributing factors include immunosuppression (chemotherapy, long-term immunosuppressive therapy), cytokine release from tumor cells, imbalance between infected and immune effector cells as well as genetic predisposition. Despite improved HLH-specific therapy (adapted components from the pediatric HLH-1994 protocol), prognosis is still poor among adult HLH patients. Due to the lack of data on adult HLH in Germany, a national multicenter registry (http://www.hlh-registry.org/) was initiated. Methods: Patients (pts) with proven or suspected HLH were registered by 35 institutions across Germany from August 2010 to July 2016. Both HLH-2004 diagnostic criteria and the HScore (www.saintantoine.aphp.fr/score/) were used to confirm HLH diagnosis. To characterize adult HLH patients, data referring to underlying disease, treatment, outcome, clinical manifestations and laboratory findings were recorded. Where available, patient samples were tested for mutations of the perforin gene PRF1 by standard Sanger sequencing. Results: A total of 125 pts (48 female) were enrolled in our registry, of whom 105 pts either fulfilled diagnostic criteria according to HLH-2004 diagnostic guidelines (n=96) or met at least 4 out of 8 criteria and reached HLH-probability of over 90 % in the HScore (n=9). Among these 105 pts, n=38 (36 %) were female and n=67 (64 %) male. Median age at diagnosis was 49 years (range 17 - 81). Trigger diseases were in line with the literature, with infections (n=34) and malignancy (n=40) being most frequent (Table 1). Patients show a wide spectrum of underlying conditions, i.e. allogeneic stem cell transplantation (alloSCT) or HLH-mimicking diseases due to cytokine release in response to blinatumomab therapy. Late onset hereditary HLH was found in 3 pts (XLP-1 and -2 respectively in EBV-coinfected pts, one pt with perforin mutation - see below). 22 of 105 patients (21 %) were tested for PRF1 mutations. A compound heterozygous PRF1 A91V/Q405X mutation was identified in one pt presenting with NK/T-cell lymphoma. Heterozygous PRF1 A91V mutations were found in 2 pts with B-cell lymphoma and HLH following alloSCT respectively, in one case with available buccal swab DNA to prove germline origin of the mutation. Table 2 summarizes clinical and laboratory findings in the cohort. A median ferritin value of 32,000 µg/L underlines the importance to evaluate pts with highly elevated ferritin with respect to potential HLH diagnosis. Apart from fever and splenomegaly, clinical presentation frequently comprised hepatomegaly, liver failure, hyperbilirubinemia, renal failure, lung involvement like ARDS, or bleeding. Treatment included steroids in the vast majority of pts (n=89), often combined with i.v. immunoglobulins (n=47) and etoposide (n=49). After a median follow up time of 164 days, 48/97 pts (49.5 %) were alive, 8 pts were lost to follow up. Survival analysis revealed median overall survival of 454 days (Figure 1a). Comparing malignancy-associated HLH and HLH after alloSCT with infection- and autoimmune-associated HLH or HLH due to unknown triggers, survival was significantly poorer in the malignancy/alloSCT group (Figure 1b). Conclusions: HLH trigger conditions in adult patients in Germany are in accordance with published case series. Outcome in adult HLH is still poor, with malignancy-associated and HLH after alloSCT showing the worst prognosis. Diagnostic vigilance and early treatment is a prerequisite for improving outcome of adult HLH. In particular, high ferritin values should raise suspicion of HLH. Disclosures Hochhaus: Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Description: The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.

Description: Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase 〉2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement–mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

Description: Quantitative determination of residual BCR-ABL transcript levels has been accepted as integral part of the management of CML patients. However, heterogeneity of molecular approaches results in a lack of comparability between different studies. Thus, there is an unmet need for harmonization of both procedures and expression of results. In a series of consensus meetings within the European LeukemiaNet a list of prerequisites to achieve an optimal sensitivity and standardization has been elaborated: use of at least 10ml peripheral blood processed within 36 hrs; bedside RNA stabilization for multicenter trials; standardized PCR protocols optimized for each platform; use of a single plasmid containing target and housekeeping genes to avoid dilution errors; use of total ABL and/or beta glucuronidase (GUS) as internal controls. To substantiate these theses an international multicenter trial within 36 labs in 14 countries was initiated. The aim of the study was to assess the variability of results obtained from different labs using the PAXgene Blood RNA System® (PreAnalytiX, Hombrechtikon, Switzerland) for RNA extraction, individual protocols for cDNA synthesis, 3 different PCR platforms (TaqMan®, TM, n=24, LightCycler®, LC, n=14, Rotorgene® n=1), and optimized quantitative RT-PCR conditions. In order to standardize results, b3a2 BCR-ABL and GUS sequences were cloned into a pCR 2.1-TOPO vector® (Invitrogen, Carlsbad, CA), which was distributed to all participants in serial dilutions as external control for quantification of BCR-ABL, total ABL, and GUS mRNA transcripts. Ten samples containing dilutions (10, 2, 1, 0.1%) of b3a2 or b2a2 BCR-ABL positive cells in normal leukocytes and negative controls were prepared, blinded, and shipped to the participants. Transcript numbers were determined in triplicates, ratios BCR-ABL/ABL and BCR-ABL/GUS were calculated and expressed in %. Median ratios BCR-ABL/ABL for b3a2 samples were 8.9, 1.7, 0.85, and 0.11%; for b2a2 samples 9.1, 1.6, 0.82, and 0.10%. Median ratios BCR-ABL/GUS for b3a2 samples were 3.4, 0.77, 0.37, and 0.042%; for b2a2 samples 2.8, 0.48, 0.29, and 0.031%. Four of 36 participants (11%) detected low BCR-ABL copy numbers in negative control samples. The coefficients of variation (CV) for all participants, TM, and LC users were 0.62, 0.57, and 0.59 for ratios BCR-ABL/ABL; 1.03, 0.85, and 1.22 for ratios BCR-ABL/GUS, respectively. Standard errors to the regression line were significantly lower evaluating ratios BCR-ABL/GUS (median 0.075, range 0.0046–0.90) compared to ratios BCR-ABL/ABL (median 0.18, range 0.022–2.2, p

Description: Abstract 862 Rapid relapse after discontinuation of imatinib, the need for indefinite therapy and residual disease in most patients are the major challenges in management of CML. Combinations of imatinib with IFN simultaneously, or consecutively preceding imatinib, or with araC may improve treatment outcome. The German CML Study Group therefore designed a randomized trial to compare standard imatinib vs. imatinib + interferon alpha (IFN) vs. imatinib + low dose araC vs. imatinib after IFN failure (for low- and intermediate-risk patients, high risk patients received imatinib 800 mg instead). The current evaluation represents the prefinal results of the pilot phase of the trial. Inclusion criteria were newly diagnosed BCR/ABL positive CML in chronic phase (CP). Primary aims are: prolongation of survival (overall, OS, and progression free, PFS), determination of rates of hematologic, cytogenetic and molecular remissions, adverse events (AE) and role of allografting. By the end of 2005, 670 patients were randomized, 13 had to be excluded (no CML (n=3), pregnancy, no CP (n=1 each), imatinib 800 mg (n=8)). Analysis was according to intention to treat. 657 patients were evaluable (174 with imatinib 400 mg, 196 with imatinib+IFN, 158 with imatinib+araC and 129 with imatinib after IFN-failure). 656 patients were evaluable for hematologic, 611 for cytogenetic, and 618 for molecular responses. Patient characteristics of treatment arms were similar for age (median 53 years), sex (40% female), median values for Hb (12.6 g/dl), WBC (66.2/μl), platelets (383/μl) and for Euro risk score (low 35%, intermediate 54%, high 10%). The median dose of imatinib was 400mg/die in all arms, of araC 10 mg per treatment day and of IFN 4.2 Mio I.U./die in the imatinib after IFN arm and 1.8 Mio I.U./die in the imatinib+IFN arm. Median observation time was 57.3 months. 55 patients died, 73 patients were transplanted in 1st CP, 81 patients progressed, 59 patients were switched to second generation TKIs. After 3 years 126 patients (72%) of the imatinib 400mg arm still received the initial therapy as well as 60 patients (30%) of the imatinib+IFN arm and 53 patients (34%) of the imatinib+araC arm. 9 patients (7%) of the imatinib after IFN arm are still on IFN. 5-year OS of all patients is 91%. 5-year PFS of all patients (no death, patient still in first chronic phase) is 87%. 5-year-OS and PFS according to treatment arm are shown in the Table. At 5 years, the cumulative incidences of achieving complete cytogenetic remission or major molecular remission (MMR) as determined by competing risks (death, progression) are not different (Table). Type and severity of adverse events (AE) over a 5-years period did not differ from those reported previously (Table). Hematologic AEs grade III/IV were similar in all therapy arms except leukopenia grade III/IV, which was more frequently observed in the imatinib after IFN arm (14%). Non hematologic AEs were mainly fluid retention, neurological and gastrointestinal symptoms and fatigue. Neurologic symptoms and fatigue were more often reported for the therapy arms with IFN. Imatinib 400mgImatinib+IFNImatinib+AraCImatinib after IFN5-Year Survival and Response RatesOS87%93%92%92% PFS84%91%88%84% CCR92%92 %89%83% MMR83%78%80%70% Adverse Events, WHO Grade III/IVAnemia7%1%3%3% Leukopenia4%5%2%14% Thrombocytopenia5%6%6%6% WHO Grade I-IVEdema15%13%5%0% Neurological5%15%5%22% Gastrointestinal17%27%21%15% Fatigue8%13%9%23% This analysis shows excellent survival and durable response rates in all arms. Currently, survival in all treatment arms is equal to, or better than in IRIS. To verify possible differences in survival, e.g. imatinib 400 mg vs. imatinib + IFN, longer observation is planned. Although cytogenetic and molecular responses in the imatinib after IFN failure arm at 5 years are inferior to that in the other treatment arms, the question of whether the consecutive therapy with IFN first and imatinib after IFN-failure provides a survival advantage requires long term follow-up. Imatinib in combination with, or after IFN, or with low dose araC are feasible and safe treatment modalities. We expect that the study will optimize and improve therapy outcome in CML. Disclosures: German CML Study Group: Deutsche Krebshilfe: Research Funding; Novartis: Research Funding; German Competence Net : Research Funding; European LeukemiaNet: Research Funding; Roche: Research Funding; Essex: Research Funding.

Description: Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases with preclinical activity against 20/21 imatinib resistant BCR-ABL mutations and clinical phase I/II efficacy in patients with chronic myelogenous leukemia (CML) and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and efficacy of dasatinib in patients (pts) with CML and ALL. We have investigated 872 peripheral blood samples from 394 pts (53% male, median age 60 yrs, range 17–85) who had been enrolled in international phase II studies investigating the activity of 70mg dasatinib BID after imatinib failure (chronic phase, CP, n=198; accelerated phase, AP, n=78; myeloid blast crisis, MyBC, n=53; lymphoid blast crisis, LyBC, or ALL, n=65). Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Hematologic and cytogenetic response data have been collected sequentially for a median of 8 months (range, 2–11) after start of therapy. Prior to dasatinib, 46 different BCR-ABL mutations involving 36 amino acids were detected in 202/394 pts (51%). 162 pts showed one, 33 pts two, 6 pts three, and 1 pt four mutations. Mutations were observed in 84 pts in CP (42%), 47 pts in AP (60%), 23 pts in MyBC (43%), and 48 pts in LyBC and ALL (74%). In patients with mutations, hematologic response was 91% in CP, 62% in AP, 41% in MyBC, and 34% in LyBC/ALL (p

Description: Caspases play a critical role in regulation of apoptosis, cell differentiation, inflammation, and innate immunity, and several are mutated or have altered expression in non-Hodgkin lymphoma (NHL). To study the impact of genetic variation in caspases on NHL risk, we analyzed tag single nucleotide polymorphisms (SNPs) in 12 caspase and related genes in 3 population-based case-control studies (1946 cases and 1808 controls). Gene-based analysis, adjusting for the number of tagSNPs genotyped in each gene, showed significant associations for CASP8, CASP9, and CASP1. SNP-based analysis showed that CASP8 rs6736233 (odds ratio (OR) CG = 1.21; ORCC = 2.13; P trend = .011); CASP9 rs4661636 (ORCT = 0.89; ORTT = 0.77; P trend = .011); and CASP1 rs1785882 (ORAT = 1.12; ORAA = 1.30; P trend = .0054) were significantly associated with NHL risk and consistent across studies. It is noteworthy that genetic variants in CASP8 were associated with risk of all major NHL subtypes. Our findings suggest that genetic variation in caspases may play an important role in lymphomagenesis.

Description: Abstract 224 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1. Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are 〉90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells. The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Light chain (AL) amyloidosis is the most common form of systemic amyloidosis and is characterized by the accumulation of aggregated, misfolded light chain protein in a variety of organs, resulting in serious organ damage and dysfunction. To date, no therapies have been approved to treat patients with AL amyloidosis, and current approaches do not directly target the underlying cause of organ dysfunction. NEOD001 is a conformation-specific antibody currently in a phase 3 clinical trial that specifically targets light chain amyloid aggregates and may promote the clearance of AL deposits by means of phagocytosis. In the present study, we compared the binding characteristics of NEOD001 to aggregates deposited in various AL organs using immunolabeling and binding assays. We also assessed the ability of the antibody to induce the clearance of AL amyloid by phagocytosis in vitro. Methods: The murine form of NEOD001, the 2A4 antibody, was used in these studies to avoid the nonspecific detection of human immunoglobulin G in human tissue. Immunohistochemical and biochemical techniques were used to characterize 2A4 immunoreactivity to AL aggregates. A total of 15 organs (heart, kidney, spleen, and liver) derived from 10 AL patients were examined. Fluorescent and chromogenic immunohistochemistry (IHC) were performed on both fresh frozen and aldehyde-fixed samples. Thioflavin T (ThioT) labeling was used to identify amyloid in IHC-labeled tissue. To assess the binding of 2A4 to AL amyloidosis patient extracts and recombinant light chain, we used surface plasmon resonance and a newly developed, plate-based immunoassay specific to AL aggregates. Phagocytosis was assessed using a macrophage cell line cultured in the presence of aggregated light chain. All experiments included isotype-control antibodies. Results: In fresh frozen sections, 2A4 demonstrated specific immunoreactivity to AL aggregates in patient samples, but not in samples from healthy subjects, for all organs examined. Although 2A4 immunolabeling largely colocalized with ThioT, additional 2A4+/ThioT-unlabeled deposits were present that likely represented amorphous light chain deposits. Immunostaining of deposits with 2A4 was almost completely attenuated in aldehyde-fixed samples, even after brief fixation (1 minute), and was minimally recovered with antigen-retrieval methods. The aggregated light chain immunoassay demonstrated 2A4 binding in all organ tissue extracts from patients with AL amyloidosis but not from healthy subjects. The specificity of 2A4 to aggregated versus monomeric light chain was confirmed by biochemical assays. 2A4 induced the rapid engagement of macrophages and the phagocytic clearance of light chain aggregate particles in vitro. Conclusions: This study demonstrates that 2A4, the murine form of NEOD001, specifically binds to amyloid light chain and amorphous light chain aggregates in various organs of patients with AL amyloidosis and, in vitro, promotes the clearance of light chain aggregate particles by macrophage phagocytosis. NEOD001 may thus hold therapeutic potential for directly targeting the underlying cause of organ dysfunction in AL amyloidosis. Disclosures Zago: Prothena Biosciences Inc: Employment, Other: Stock. Renz:Prothena Biosciences Inc: Employment, Other: Stock. Torres:Prothena Biosciences Inc: Employment, Other: Stock. Dolan:Prothena Biosciences Inc: Employment, Other: Stock. Barbour:Prothena Biosciences Inc: Employment, Other: Stock. Salmans:Prothena Biosciences Inc: Employment, Other: Stock. Shughrue:Prothena Biosciences Inc: Employment, Other: Stock. Schenk:Prothena Biosciences Inc: Employment, Other: Stock. Kinney:Prothena Biosciences Inc: Employment, Other: Stock.

Description: Acute Promyelocytic Leukemia (APL) accounts for 5% of all cases of acute myeloid leukemia (AML). This disease is highly curable with all-trans-retinoic acid (ATRA) based therapy. In non-APL AML, ATRA has limited activity, and little is known about mechanisms of ATRA resistance. The apparent selective efficacy of ATRA in PML/RARα-associated APL poses an important question as to whether the presence of this fusion protein renders APL uniquely susceptible. Two compelling arguments can be made to counter this view. First, experiments in vitro show that ATRA effectively differentiates HL-60 cell lines, which lack the PML/RARα fusion protein. Second, clinical studies with ATRA in previously untreated older AML patients (excluding APL) have reported clinical activity. These observations confirm the therapeutic potential of ATRA beyond APL. In this context, our group has previously identified the lysine demethylase LSD-1, as a therapeutic target to re-sensitize leukemic blasts to ATRA. A clinical investigation of ATRA combined with LSD-1 inhibition is currently underway (NCT02273102). It is likely that other defects leading to ATRA resistance will be similarly amenable to pharmacologic manipulation. Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of AML. Over-expression of c-Myc in leukemic blasts enhances clonogenic survival and blocks ATRA induced differentiation. We hypothesized that down-regulation of c-Myc might increase the anti-leukemic effects of ATRA in AML. To date, c-Myc has been an evasive target for direct pharmacologic inhibition however, inhibitors of the PI3K/AKT/mTOR pathway have been shown to indirectly lower levels of c-Myc in leukemic blasts. In the current study, we show that the pro-differentiation effects of ATRA are markedly potentiated when combined with agents that target PI3K/AKT/mTOR signalling. In AML cell lines and primary patient samples, we observed additive pro-differentiation effects when ATRA was combined with inhibitors of PI3K (ZSTK474) and mTOR complex proteins (Torin-1, WYE-125132). However, when combined with the bromodomain inhibitor NVP-BEZ235, a dual inhibitor of PI3K and mTOR, we observed synergistic induction of CD11b by FACS analysis. Combination studies revealed loss of cell viability, cell cycle arrest in G1 phase, and impaired clonogenic survival, which was more prominent for ATRA combination treatments than with any agent used alone (Figure 1). To assess the role of c-Myc in mediating these effects, we measured c-Myc protein levels and PI3K/AKt/mTOR pathway markers at different time-points following treatment with ATRA alone and in combination with the inhibitors described above (Figure 2). Our findings suggest that ATRA alone quickly down-regulates c-Myc (within 6 hours) through transcriptional repression. Disruption of the PI3K/AKT/mTOR pathway further down-regulates c-Myc (within 3 hours) through destabilization and enhanced degradation. ATRA combined with NVP-BEZ235 produced maximal c-Myc suppression, and led to more cell kill than any other combination tested. Detailed analysis of changes in the transcriptome in MV-411 cells following treatment with ATRA and NVP-BEZ235 revealed that both agents act jointly on the regulation of the same biological pathways and processes, but regulate different sets of genes within these pathways. Updated mechanism based studies will be presented. In conclusion, suppression of c-Myc levels through disruption of PI3K/AKT/mTOR signalling augments the anti-leukemic effects of ATRA. These data support the clinical investigation of ATRA combined with rapalogs or bromodomain inhibitors. Figure 1. Combination treatment with PI3K/mTORC inhibitors and ATRA decreases cell viability in AML cells. MV4-11 cells were treated as indicated with combinations of BEZ (1µM), WYE (1µM) or ZSTK (2.5µM) and ATRA (0.1 µM). Number of cells was determined by CellTiter-Glo¨ luminescent cell viability assay (Promega). Data were analyzed by one-way ANOVA (P 〈 0.0001) followed by TukeyÕs post-hoc test. * P〈 0.05, ** P 〈 0.01, *** P 〈 0.001,**** P 〈 0.0001. Figure 1. Combination treatment with PI3K/mTORC inhibitors and ATRA decreases cell viability in AML cells. MV4-11 cells were treated as indicated with combinations of BEZ (1µM), WYE (1µM) or ZSTK (2.5µM) and ATRA (0.1 µM). Number of cells was determined by CellTiter-Glo¨ luminescent cell viability assay (Promega). Data were analyzed by one-way ANOVA (P 〈 0.0001) followed by TukeyÕs post-hoc test. * P〈 0.05, ** P 〈 0.01, *** P 〈 0.001,**** P 〈 0.0001. Figure 2. Reduced expression of MYC protein by inhibition of the PI3K/AKT/mTORC pathways. Immunoblotting/quantification of MYC protein levels in MV4-11 cells following treatment with combinations of WYE (1µM), BEZ (1µM), ZSTK (2.5µM) and ATRA (0.1 µM). Figure 2. Reduced expression of MYC protein by inhibition of the PI3K/AKT/mTORC pathways. Immunoblotting/quantification of MYC protein levels in MV4-11 cells following treatment with combinations of WYE (1µM), BEZ (1µM), ZSTK (2.5µM) and ATRA (0.1 µM). Disclosures No relevant conflicts of interest to declare.