ALBERT

Description: Activating mutations in Flt3, N- and K-Ras have been reported in all AML subtypes and represent common molecular defects in de novo AML. We have previously shown that these mutations lead to constitutive AKT phosphorylation and activation. As a consequence, Akt phosphorylation is found in myeloid blasts of the majority of AML patients. We reasoned that constitutively active AKT may contribute to leukemia development, and therefore we assessed the contribution of AKT in oncogenic transformation in vivo. For this purpose, we established an inducible mouse model expressing myristylated AKT1 under the control of the scl-3′ enhancer (MyrAKT1). This system restricts activated AKT1 to endothelium, hematopoietic stem cells and myeloid lineage cells at a low but detectable level. About 40% of induced mice developed a myeloproliferative disorder after latencies of 7 to 22 months. Onset of disease was frequently associated with hemangioma formation, due to endothelial MyrAKT1 expression. The myeloproliferative disorder was associated with splenomegaly with increased extramedullary hematopoiesis, while the peripheral blood contained mature granulocytes. Furthermore, the stem cell and progenitor cell compartment in spleens and bone marrow of these mice was altered compared to control mice. Colony formation assays with MyrAKT1-expressing bone marrow suggested that overactivation of AKT1 enhanced proliferation. The AKT1-induced disease was transplantable by both bone marrow and spleen cells. These findings highlight the oncogenic capacity of constitutively activated AKT1 in vivo and indicate that AKT is an attractive target for therapeutic intervention in AML.

Description: Key Points GlycoPEGylated FVIII (N8-GP) demonstrates the same efficacy and prolonged effect in animal models as native FVIII. Circulatory half-life of glycoPEGylated FVIII (N8-GP) is prolonged by approximately twofold in several species.

Description: Key Points Juvenile zebrafish tolerate widespread coagulopathy due to complete ablation of antithrombin III, but develop lethal thrombosis as adults. In vivo structure/function analysis of antithrombin III in zebrafish reveals limited roles for heparin-binding and anti-IXa/Xa activity.

Description: Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLB megaTAL resulted in 〉90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLB disruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLB gene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLB megaTAL-treated CAR T cells in vivo. While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLB megaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLB knockout enhances the potency of CAR T cells. In summary, the CBLB megaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitro and in vivo, and thus could potentially improve the efficacy of CAR T therapy. Disclosures Hooper: bluebird bio: Employment, Equity Ownership. Havens:bluebird bio: Employment, Equity Ownership. Krostag:bluebird bio: Employment, Equity Ownership. Magee:bluebird bio: Employment, Equity Ownership. Martin:bluebird bio: Employment, Equity Ownership. Gupta:bluebird bio: Employment, Equity Ownership. Smurnyy:bluebird bio: Employment, Equity Ownership. Pechilis:bluebird bio: Employment, Equity Ownership. Rode:bluebird bio: Employment, Equity Ownership. Chavkin:bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership.

Description: Introduction Turoctocog alfa pegol (N8-GP) is a glycoPEGylated recombinant factor VIII (rFVIII) under development for treatment of hemophilia A (HA). Patients with severe HA receiving intravenous (IV) replacement therapy have 20-40% risk of developing inhibitory FVIII antibodies. It is unclear whether the absence of inhibitors in some patients is due to lack of response or due to peripheral tolerance. Induction of peripheral tolerance may be dependent on regulatory T-cells (Tregs) since they are suggested to be involved in regulation of the anti-FVIII antibody response. In this study, tolerance induction following IV rFVIII administration was assessed in humanized HA mice, described as a suitable model for tolerance induction as only a fraction produce anti-FVIII antibodies after IV FVIII exposure (Steinitz KN, et al. Blood 2012;119(17):4073-4082). Aims To explore whether IV exposure to rFVIII induces peripheral tolerance to subcutaneous N8-GP (SC N8-GP) exposure in humanized HA mice, and whether Tregs play a role in tolerance induction. Methods HLA-DR transgenic FVIII-deficient mice lacking murine MHC class II were treated with rFVIII IV once per week for 8 weeks, then challenged with SC N8-GP once a week for 8 weeks. The role of CD25+ Tregs was studied by treating one group of mice with depleting anti-CD25 antibodies during the IV exposure period. The presence of anti-FVIII antibodies was measured prior to IV and SC dosing, and at study end. Mice with anti-FVIII antibodies after IV rFVIII exposure were excluded from the analysis. Results No anti-FVIII antibodies were detected after challenge with SC N8-GP in mice pretreated with IV rFVIII. Depletion of CD25+ Tregs (confirmed by flow cytometry) during the IV exposure did not significantly affect the anti-FVIII antibody response. Conclusion It was demonstrated that peripheral tolerance to SC N8-GP was induced in humanized HA mice not responding to IV rFVIII exposure. Depletion of CD25+ Tregs during IV exposure was insufficient to abolish the induction of tolerance. Disclosures Reedtz-Runge: Novo Nordisk A/S: Employment. Weldingh:Novo Nordisk A/S: Employment. Schiøler Schultz:Novo Nordisk A/S: Employment. Holm Millner:Novo Nordisk A/S: Employment. Rode:Novo Nordisk A/S: Employment. Wiinberg:Novo Nordisk A/S: Employment. Kjelgaard-Hansen:Novo Nordisk A/S: Employment.

Description: Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, a tyrosine kinase receptor. The signalling pathways and downstream targets are only partly understood. We demonstrated that the presence of Flt3-ITD constitutively activated Akt (PKB), a key serine-threonine kinase within the PI3-kinase pathway. The constitutive activation of Akt induced phosphorylation and transcriptional inhibition of the transcription factor Foxo3a. Also, forced Foxo3a activity reversed Flt3-ITD-mediated growth properties. Using a novel Akt inhibitor, we showed that Flt3-ITD-mediated Foxo3a phosphorylation was dependent on Akt. Conditional Akt activation targeted to the cell membrane conferred cytokine-independent survival, proliferation and transformation of myeloid progenitor cells. Induction of Akt activity in vivo promoted the development of a myeloproliferative disease in a syngeneic mouse model. Importantly, phosphorylation of Akt was frequently found in primary myeloid blasts, supporting that this signalling pathway may be important in the development of AML. Furthermore, activation of Akt constitutively activated the downstream kinase mammalian target of rapamycin (mTOR). mTOR has several important biological functions, notably in nutrient sensing and protein translation. Treatment with rapamycin, a specific inhibitor of mTOR, inhibited mTOR kinase activity at 1nM in all examined cell systems. Rapamycin caused cell cycle arrest, apoptosis and completely inhibited transformation in FLT3-ITD-positive cells. These effects were most pronounced in the low nanomolar range, which are therapeutic concentrations of rapamycin-treated patients. The biological advantage in Akt-expressing cells was fully reversed by treatment with rapamycin. In all examined primary blasts from AML patients, mTOR was inhibited by rapamycin. In short term proliferation assays, twelve out of 18 (67%) examined showed marked sensitivity with an IC50 below 10nM rapamycin, while one third were found to be more resistant. Taken together, increased survival, proliferation and leukemic transformation by Flt3-ITD is mediated by Akt and mTOR, and can be inhibited by rapamycin. We conclude that rapamycin may be an attractive substance for the treatment of patients with AML.

Description: The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

Description: Introduction: Due to its heterogenous nature including evolving mutations, multiple myeloma (MM) is still an incurable disease. Several novel agents have been introduced during the last decade resulting in prolonged median overall survival (OS) up to 7-8 years. Nevertheless, disease relapse is expected and eventually all patients will end up with relapsed and refractory multiple myeloma (RRMM). To benefit from all available drugs, novel combinations should be evaluated for feasibility and efficacy. Bortezomib (B), elotuzumab and dexamethasone (d) showed superiority to Bd with progression-free survival (PFS) of 9.7 vs. 6.9 months, respectively, without excessive toxicity. In this study we investigated the safety and efficacy of carfilzomib (K), elotuzumab (E) and dexamethasone (D) combination (KED) in RRMM patients. Patients and methods: Altogether 15 RRMM patients after 1-3 prior lines including B or/and lenalidomide (Len) were included. Refractoriness for B or/and Len was allowed. The main exclusion criteria were age 〉 74 years, absolute neutrophil count 〈 1.0 x 109/L and platelet count 〈 75 x 109/L, hemoglobin 〈 80 g/L, active heart disease or left ventricular ejection fraction 〈 40%. Carfilzomib was given once weekly 20 mg/m2 on C1D1 and thereafter 70 mg/m2 in 28-day cycles on day 1, 8, 15 in cycles 1-8, thereafter on day 1, 15 combined with weekly elotuzumab 10 mg/kg on day 1, 8, 15 in cycles 1-2, thereafter on day 1, 15; dexamethasone 40 mg on day 1, 8, 15, 22 in cycles 1-8, thereafter on day 1, 15. Treatment continued until progression (PD) or toxicity. The primary endpoint was overall response rate (ORR). If patients achieved at least very good partial remission (VGPR) the quality of response was assessed with high-sensitivity multicolour flow cytometry according to the 8-color EuroFlow protocol. Patients also completed health-related quality of life (QoL) questionnaires at day 1 and 15 through cycle 1-8. Results: The median age of patients was 69 (62-73) years, 80% (12/15) had received upfront autologous stem cell transplantation. The median number of prior lines was 2 (1-3). Proportion of Len or B refractory was 47% and 20% were double refractory. FISH, beside at diagnosis, was repeated and 73% (11/15) had at least one of following aberrations: del17p, t(4;14), t(14;16), t(14;20) or +1q. Del17 was found in 27% (4/15) of patients and +1q was frequent (67%). After the median follow-up of 18 months the ORR (at least partial response, PR) was 87% (13/15). Seven patients (47%) achieved VGPR, of these 6 patients continue in study, one was withdrawn due to thrombotic microangiopathy (TMA). The median measurable MRD level of VGPR patients was 0.005% (range 0.002-0.15%, one undetectable), and 0.003% (range 0.0007-0.26%) after one year, respectively. Regarding the rest of the patients 6 achieved PR, one minimal response and one was refractory. The median time to response was 7 (3-16) weeks. The estimated median PFS is 22 months (CI 95% 17.4-27.1). The OS has not been reached. Two (13%) grade 2 infusion reactions during first infusion were noticed. One patient developed autoimmune hemolytic anemia (AIHA), but recovered and continued KD without reappearance of AIHA. Grade 1-2 respiratory infections appeared in 66% of patients. Altogether 22 severe adverse events (SAE) grade 3-4 were reported in 60% (9/15) patients, one third were infections. Grade 3-4 neutropenia was found in 40% which is slightly more compared to reports when elotuzumab is combined with Len or pomalidomide. The overall QoL trajectory was kept steady throughout the 8 cycles. Diarrhea and dyspnoe were reported during treatment, but with full recovery before starting the next cycle which had no consequence for further treatment. Conclusion: The primary endpoint, at least PR, was reached in 87% of study patients. After a median follow-up of 18 months KED resulted in durable responses in 7 of these 15 patients (47%), despite the high frequency of adverse cytogenetics by FISH, 67% with +1q and 27% with del17p. The number of non-hematological SAEs was 60%. We noticed 2 serious unexpected adverse reactions, AIHA and TMA with convulsions, and an additional case of pulmonary embolism, in responding patients. We recommend careful monitoring for the signs of microangiopathy and hemolysis. In summary, KED resulted in a high ORR, which was durable in approximately 50% of the patients. Side effects were manageable and randomized trials with KED are warranted. Disclosures Silvennoinen: Cancer patients Finland: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Anttila:Janssen: Honoraria, Other: Advisory Board; BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Lievonen:Amgen, Celgene, BMS, Sanofi, Takeda: Consultancy; Celgene, Janssen, Novartis, Amgen: Honoraria; Cancer Association Finland: Honoraria. Varmavuo:Abbvie, Roche, Celgene, Amgen, Sanofi: Consultancy. Säily:Boehringer Ingelheim: Honoraria; Janssen Cilag: Honoraria; Takeda: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Roche: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Partanen:Takeda: Other: Scientific Advisory Board Meeting; Behring: Honoraria; Abbvie: Honoraria, Other: Scientific Advisory Board Meeting. Sankelo:Celgene, Amgen, Sanofi: Other: Congress travel support. Luoma:Amgen, Janssen. Incyte: Other: Advisory Boards. Jantunen:Celgene: Honoraria; Amgen: Honoraria, Other: Advisory Board; Sanofi: Honoraria; TEVA: Other: Advisory Board; Takeda: Other: Advisory Board. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.