ALBERT

Description: Since the molecular cloning of thrombopoietin (TPO), two types of recombinant human TPO, full-length recombinant human TPO (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF), have been tested in a variety of clinical trials. The agents showed clinical efficacy in some cases. However, neutralizing antibodies against endogenous TPO were generated in some healthy volunteers who received PEG-rhMGDF, resulting in thrombocytopenia. Recently, several TPO mimetics are being developed, such as peptide-based molecules, nonpeptidyl small compounds and agonist antibodies. Here we report generation of novel fully human anti-human Mpl agonist antibodies. Two lead monoclonal antibodies MA01 and MA02 were cloned from human antibody-producing mice (KM mice™) immunized with human Mpl-expressing cells. Both MA01 and MA02 were of immunoglobulin gamma 1 (IgG1). These IgG1 agonists promoted proliferation of UT7/TPO cells in vitro, but were 〉100-fold less effective than TPO. We, therefore, genetically modified MA01 and MA02 in order to enhance the activity and to minimize the effector function. First, constant heavy (CH) region of MA01 was converted to IgG4 (MA01G4) to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This conversion affected neither binding affinity nor agonistic activity of MA01. Next, hinge region of MA01G4 was changed to human IgG3 hinge (MA01G4344). MA01G4344 showed no change in binding affinity, but showed 10-fold higher activity than original MA01. In the case of MA02, the CH region was converted from G1 to G3344 to generate MA02G3344, in which CH1-hinge region was of IgG3 and CH2-CH3 region was of IgG4. The conversion drastically improved agonistic activity. Although MA02 (G1) activated Jak2 and Erk1/2 but not STAT5 and Akt, MA02G3344 activated all of them. These data suggest that conversion of CH1-hinge region to IgG3 type is essential for agonistic activity of anti-Mpl antibodies. The effective concentration 50 (EC50) values of MA01G4344 and MA02G3344 in UT7/TPO assay were in the range of 0.01–0.1nM. These antibodies stimulated human cord blood-derived CD34+ cells to give rise to CFU-MK colonies without any other supportive cytokines. A single injection of MA01G4344 increased platelet count of human Mpl transgenic mice for over one month. These domain subclass-converted antibodies are expected to provide a new medical option for treatment of chronic thrombocytopenia without losing good properties of natural antibody such as long serum half-life and low immunogenicity. Figure Figure

Description: Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly, high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However, mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here, we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow, which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model, Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore, we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation, which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1.

Description: Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

Description: Abstract 1962 Poster Board I-985 Introduction: AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results: To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions: We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures: No relevant conflicts of interest to declare.

Description: Human interleukin-3 receptor alpha (IL-3Ra, CD123), which promotes the proliferation and differentiation of hematopoietic cells, is highly expressed in myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We newly generated KHK2823, a non-fucosylated fully human IgG1 monoclonal antibody against human IL-3Ra, by utilizing the POTELLIGENT® technology. Here, we describe the in vitro and in vivo preclinical efficacy and safety of KHK2823, as well as its pharmacodynamic (PD) profile. At first, we explored that KHK2823 bound to various hematological malignant cells and leukemic stem cells. The cells from AML and MDS bone marrows were found to be bound by KHK2823. A significant part of bone marrow cells derived from B-cell acute lymphoblastic leukemia (B-ALL) patients was also bound by KHK2823. KHK2823 bound to soluble human IL-3Ra protein with a sub-nanomolar dissociation constant (KD), and recognized CD34+ CD38+ (leukemic blast) and/or CD34+ CD38- (leukemic stem cell) cells in patients with AML/MDS, as well as AML cell lines, thereby obtaining a high antibody-dependent cellular cytotoxic activity without complement-dependent cytotoxicity. Interestingly, KHK2823 did not interfere with the binding of IL-3 to IL-3R. The lack of a receptor-ligand interaction may conserve the IL-3 signal, which plays an important role in normal hematopoiesis. In a tumor model xenografting the human AML cell line MOLM-13 on nude rats, KHK2823 significantly suppressed the tumor growth at doses of 0.1 and 1 mg/kg (Figure 1). The PD and toxicity profiles of KHK2823 were assessed in cynomolgus monkeys administered at doses ranging from 0.1 to 100 mg/kg by i.v. infusion, once weekly for 4 weeks. KHK2823 was generally well tolerated in monkeys, even at 100 mg/kg. The number of IL-3Ra-positive cells in the peripheral blood of cynomolgus monkeys decreased in all groups receiving KHK2823, which suggest KHK2823 could exert its depletion activity of IL-3Ra-positive cells in human (Figure 2). Currently, the safety and tolerability of KHK2823 is being investigated in patients with AML or MDS in a Phase 1 study (NCT02181699, https://clinicaltrials.gov/ct2/show/NCT02181699). This is the first non-randomized, open-label, dose escalation clinical study to investigate the safety, PK, immunogenicity and PD of repeated doses of KHK2823. In summary, KHK2823 was confirmed to bind to AML, MDS and B-ALL cells as the IL-3Ra in accordance with other publications. KHK2823 was also found to eliminate AML cells in vitro and also suppressed the AML tumor growth in the in vivo model. In addition, the number of IL-3Ra-positive cells in cynomolgus monkeys decreased following i.v. infusion of 0.1mg/kg KHK2823 with a tolerable safety profile, even at a dose of 100 mg/kg. Taken together, KHK2823 may therefore be a promising anti-IL-3Ra therapeutic drug for the treatment of AML. Figure 1. Antitumor activity of KHK2823 in a tumor xenograft nude rat model Figure 1. Antitumor activity of KHK2823 in a tumor xenograft nude rat model Figure 2. PD profile of KHK2823 in cynomolgus monkeys Figure 2. PD profile of KHK2823 in cynomolgus monkeys Disclosures Akiyama: Kyowa Hakko Kirin Co., Ltd.: Employment. Takayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment. Maekawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Shimabe:Kyowa Hakko Kirin Co., Ltd.: Employment. Nishikawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Yamawaki:Kyowa Hakko Kirin Co., Ltd: Employment. Iijima:Kyowa Hakko Kirin Co., Ltd: Employment. Hiura:Kyowa Hakko Kirin Co., Ltd.: Employment. Takahashi:Kyowa Hakko Kirin Co., Ltd.: Employment. Akashi:Asahi Kasei: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau. Tawara:Kyowa Hakko Kirin Co., Ltd: Employment.

Description: The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in murine leukemia. In humans, Evi-1 is located on chromosome 3q26, and rearrangements on chrmosome 3q26 often activate Evi-1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Patients with these karyotypes are characterized by the elevated platelet count and lack of response to antileukemic therapy. Elevated Evi-1 expression occurs with high frequency in AML patients without 3q26 abnormalities, and is also associated with unfavorable outcomes. Thus, Evi-1 is one of the key factors that predict poor survival in leukemia patients. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains a total of 10 zinc finger motifs organized in two discrete domains, comprising 7 and 3 repeats respectively, which have distinct DNA binding specificities. Recently, we generated Evi-1-mutant mice and showed that Evi-1 plays an essential role in proliferation of both hematopoietic stem cells (HSCs) and transformed leukemic cells. Furthermore, we identified candidate target genes of Evi-1 using gene expression profiling analysis in HSCs combined with the gene expression data of AML samples. These genes include Gata1, Gata2, Angpt1, Mpl, Jag2, Pbx-1, Setbp1 and Itga2b. In this study, we first analyzed relative gene expression of these candidate genes in control- or Evi-1-transduced hematopoietic stem/progenitors (c-Kit+ cells). Among these candidate genes, Evi-1 up-regulates transcription of Pbx-1, Mpl, Setbp1 and Itga2b. Next, we cloned 5 ′ up-stream genomic regions of these four genes into the pGL-4 luciferase reporter vector, and found that Evi-1 increased luciferase activity of Pbx-1 reporter construct in COS7 cells. Deletion of reporter constructs revealed that Evi-1 binds to -0.5kb upstream of the transcription start site of Pbx-1. We then examined the transcription activity of a series of Evi-1 mutants and found that both the first and second zinc finger domains are required for the Pbx-1 up-regulation. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that Evi-1 directly binds to the promoter region of Pbx-1. We next evaluated a role for Pbx-1 in Evi-1-induced myeloid transformation. Bone marrow progenitors transduced with Evi-1 showed sustained colony formation in the serial replating assay. After establishment of sustained clonogenic activity following more than three rounds of replating in methylcellulose medium, the cells were transduced with control or Pbx-1-shRNA. Interestingly, reduction of Pbx-1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx-1 did not impair bone marrow transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF), suggesting that Pbx-1 is specifically, as opposed to generally, required for maintenance of transformation mediated by Evi-1. Taken together, these results indicate that Pbx-1 is one of the direct target genes of Evi- 1 in hematopoietic cells, and aberrant Pbx-1 expression is responsible, at least in part, for the oncogenic activity of Evi-1. Because Pbx-1 is known as a critical regulator of hematopoietic stem cells and leukemia development, the Evi-1-Pbx-1 pathway may be a key modulator of stem cell activity in normal and malignant hematopoiesis.

Description: Evi-1 (ecotropic viral integration site-1) is a nuclear transcription factor containing multiple zinc finger motifs, and plays an essential role in the proliferation/maintenance of hematopoietic stem cells. Aberrant expression of Evi-1 has been frequently found in myeloid leukemia as well as in several solid tumors, and is associated with a poor patient survival. It has been shown that Evi-1 acts as a transcriptional repressor through its interaction with several transcriptional regulators, including C-terminal binding protein (CtBP), histone deacetylases (HDACs), and Smad3. Numerous studies have shown that cancer cells are characterized by prominent epigenetic dysregulation, including histone modifications. Methylation of histone H3 lysine 9 (H3K9) is one of the most well-studied histone modifications. After the initial identification of SUV39H1 as a H3K9-specific histone methyltransferase (HMT), at least three other HMTs, G9a, GLP, and SETDB1, have been recognized as HMTs for H3K9 in mammals. Very recently, several groups reported that Evi-1 physically interacts with H3K9 HMTs, SUV39H1 and G9a. However, the functional roles of the HMTs in Evi-1-mediated leukemogenesis remain unclear. In this study, we first showed that Evi-1 physically interacts with SUV39H1 and G9a in 293T cells using immunoprecipitation experiments. Immunofluorescence analysis also revealed that Evi-1 co-localizes with these HMTs in COS7 cells. Thus, Evi-1 forms a complex with these HMTs in vivo. We then attempted to map the region of Evi-1 that is necessary for interaction with the HMTs using Evi-1 deletion mutants which lack various functional domains. In contrast to the previous reports, all of these deletion mutants associate with both SUV39H1 and G9a almost as efficiently as full-length Evi-1, suggesting that interaction between Evi-1 and the HMTs is mediated through a relatively wide stretch of multiple regions. We further showed that both Evi-1-SUV39H1 and Evi-1-G9a complexes are able to methylate recombinant H3 using in vitro histone methylation assay, suggesting that the proteins form an active complex with methyltransferase activity. Next, we performed a luciferase reporter assay to determine whether the HMTs are actively involved in Evi-1-mediated transcriptional repression of the p3TP promoter, which is induced strongly by TGF-β. As we have shown previously, co-transfection of Evi-1 together with p3TP-Lux resulted in repression of reporter activity. Interestingly, catalytically inactive forms of SUV39H1 and G9a, carrying a point mutation within the HMT domain, were able to abrogate the transcriptional repression mediated by Evi-1. Because the inactive mutants were still able to associate with Evi-1 in immunoprecipitation studies, they might act as dominant-negative mutants, competing with endogenous HMTs. Finally, we evaluated a role for the HMTs in Evi-1-induced myeloid transformation. Bone marrow progenitors transduced with Evi-1 showed sustained colony formation in the serial replating assay. After establishment of sustained clonogenic activity following more than three rounds of replating in methylcellulose medium, the cells were transduced with SUV39H1-shRNA, G9a-shRNA or control-shRNA. Remarkably, RNAi-based knockdown of these HMTs in Evi-1-transformed progenitors markedly reduced their colony-forming activity. Taken together, these results indicate that Evi-1 can act as a transcriptional regulator that is able to form higher order complexes with HMTs, and this association has a role in the transcription repression and leukemia development. Therefore, epigenetic modifications mediated by SUV39H1 and G9a could be valid therapeutic targets in Evi-1-related hematological malignancies.

Description: Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL–mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL–immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell–like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell–derived MLL leukemic cells.