Human Acute Myelogenous Leukemia Research Articles

Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager.

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.

Biochips to measure ultraviolet light absorption by leukemia cells

During the treatment of leukemia, side effects create a great burden for patients. Therefore, new therapies to replace conventional chemotherapy and hematopoietic stem cell transplantation are highly desirable. In a previous study, I found that UV irradiation at 365 nm induced apoptosis in 98% of leukemia (Jurkat T) cells. To determine the most suitable wavelength for such treatments, it is necessary to measure the UV light absorption of cancer cells. In this study, a biochip was fabricated and evaluated for this purpose. Three types of leukemic cells (human acute myelogenous leukemia cells, human acute lymphoblastic leukemia cells, and human chronic myelogenous leukemia cells) were injected into the biochip, and light absorption was measured, revealing that individual leukemic cells have different light absorption characteristics. This variation is important in order to develop an implant care device to treat leukemia more effectively.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients

BackgroundMCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902).MethodsBinding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy.ResultsHere we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings.ConclusionsThe selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

The RNA m6A Reader YTHDF1 Is Required for Acute Myeloid Leukemia Progression.

The m6A reader YTHDF1 is required for progression of acute myelogenous leukemia and can be targeted with the FDA-approved drug tegaserod to suppress leukemia growth.

Specific Nucleotide Substitutions and Stressors Erode Highly Conserved Genomic Regions in Acute Myelogenous Leukemia

Background: "Leukemia stressors” refer to extrinsic factors that may facilitate clonal progression. We previously reported that smoking initiates unique leukemogenic signatures in acute myelogenous leukemia (AML) characterized by a higher proportion of C>A and G>A single base substitutions (SBS) [Hoang. ASH. 2021]. These G>A SBS are known to be particularly prevalent in CpG islands due to cytosine methylation. However, toxic smoking metabolites could potentially induce specific genomic signatures in patients with AML that resemble COSMIC SBS4 in lung cancer [i.e. enrichment in C>A transversion], and spontaneous C>T SBS1 signatures within specific genomic clusters or nodes. In this study, we investigate how these SBS1/SBS4 signatures distribute within hot-spot regions and evaluate whether G>A mutations (mut), commonly observed in methylated CpG islands, are differentially expressed in AML patients who are older and/or are smokers. In our study, these hot-spot regions or nodes were referenced as a genomic pool 1 (GP1). Methods: After IRB approval, the incidence of specific SBS densities were investigated in vulnerable hot-spot regions affected by exogenous [SBS4] and endogenous [SBS1] mutagenesis. Next generation sequencing (NGS) was used in patients to obtain data regarding specific mutation types and locations. In known gene exons, we examined genomic coordinates corresponding to individual mut. COSMIC browser allowed genomic coordinate examination to "aggregate” mut within putative "vulnerable exon neighborhoods". "Hot-spot neighborhoods” were described as genomic pool 1 (GP1). Descriptive and inferential statistics were used to analyze data, which was obtained from a single institution. Results: 80 patients (pt) diagnosed with hematopoietic malignancies with available next generation sequencing (NGS) were selected for analysis. 73/80 (91%) were AML pt. The median age was 63 years (y) (range, 22-88). 42% and 64% of pt were smokers and older than 63 y, respectively. With respect to specific hot spots or nodes, AML pt showed increased G>A mutagenicity in GP1 cluster 1 (Fig 1). Specifically, for G>A, smoking increased the rate from 18.4% to 37.5% (p=0.01) (Fig 1 A and B). Age > 63 y led to an increased mutagenicity rate of 17.1% to 30.5% (p=0.03) (Fig 1 C and D). A synergistic effect in mutagenicity was observed when combining age > 63 y plus smoking (10.3% to 41.9%, p=0.01) (Fig 1. E and F). In regards to hotspots in specific genes, smoking has the ability to increase NRAS (G>A), P53 (G>A) and SRSF2 (C>A) frequencies within GP1. Interestingly, smoking increased C>A mutagenicity in the SRSF2 gene in GP1 from 50% vs 82%. Conclusions: Our data suggests that aging and smoking globally and locally erode the AML genome by potentially accelerating cytosine methylation and thus G>A mutagenicity, most likely in CpG islands within genes like NRAS and P53. These genes likely contain hot spots aggregated in GP1 that, when mutated, can de-stabilize the genome and increase the risk of AML. Importantly, we observed similar COSMIC SBS4 substitutions seen in lung cancer associated with C>A transversions in older AML pt who had a history of smoking, which is specifically found in the SRSF2 gene. Overall, our data advances our understanding on how risk factors for AML affect the human AML genome. These have strong implications for AML prevention, pathogenesis and therapy. Future studies should be aimed at identifying other intrinsic and extrinsic risk factors for AML seeking to explore their specific mutational patterns and locations within the human genome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combinatorial Anti-Leukemic Activity Of Liposomal Ceramide and Inhibitors Of Autophagy

BackgroundCeramides are naturally occurring cellular sphingolipids which are increased following various effective cancer therapies. Bioavailable forms of ceramide are under development as therapeutic tools (Anticancer Agents Med Chem. 2011 Nov;11(9):911-9). Short side chain (C6) ceramide has, in pegylated nanoliposomal (NL) formulations, shown excellent cellular uptake and significant anti-tumor activity in a variety of tumor types and models. Cellular autophagy is an active microsomal process implicated in ceramide metabolism and potentially in the detoxification of C6-ceramide. We have previously shown synergistic combination therapy with Lip-C6-ceramide and vinblastine and its association with autophagy dysfunction in hepatocarcinoma and colorectal cancer models (Cancer Lett. 2013 Sep 1;337(2):254-65.). Methods and findingsWe explored the therapeutic activity of nano-liposomally delivered C6-ceramide (Lip-C6) in cell lines and primary human acute myelogenous leukemia (hAML). Lip-C6 cytotoxicity was tested in multiple human and murine AML lines (in short term viability assays) and in multiple cryopreserved primary human AML cases (in clonogenic assays - colony formation in methylcellulose). Lip-C6 demonstrated anti-AML activity in these cells with an EC50 of 2-23uM Lip-C6. In order to enhance this efficacy the interplay between ceramide induced apoptosis and autophagy was explored. The AML line KG1 was found to be relatively resistant to Lip-C6 (EC50 23uM). KGI showed supra-additive sensitivity to the combination of Lip-C6 with either vinblastine (VBL) or vincristine, both microtubule binding agents with the ability to reduce autophageic flux. KG1 sub-clones with knockdown of ATG5 demonstrated increased sensitivity to Lip-C6 relative to control or scrambled knockdown transfectants, consistent with the hypothesis that autophagy “rescues” this line from ceramide cytotoxicity. To further confirm these findings we examined the interaction of Lip-C6 (20uM) with VBL (5uM) or chloroquine (25uM) in a series of 13 fresh, patient derived AML cases (Figure: A-F). After 48 hours of co-culture, cells were examined for apoptosis of whole leukemic(E) and putative leukemia “stem cell” (LSC - F) fractions by flow cytometry. We also studied the in-vivo activity of Ghost (control) NLs, Lip-C6, VBL-NLs and combinatorial Lip-C6+VBL-NLs in NSG xenografts bearing a single poor prognosis (inversion 3) hAML (data are shown in the figure G.) [Display omitted] ConclusionsWe conclude that VBL and chloroquine both yield significant enhancement of Lip-C6 ceramide anti-hAML cytotoxicity. Available evidence suggests that this effect is mediated by inhibition of autophagy in these ceramide stressed cells. These data suggest that the combination of Lip-6 with autophagy inhibitors delivered simultaneously or in combinatorial formulations may prove clinically useful for these disorders. Disclosures:Kester:Keystone Nano Inc.: Membership on an entity's Board of Directors or advisory committees, Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano., Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano. Patents & Royalties.

IQGAP1: A Potential Therapeutic Target in Acute Myelogenous Leukemia (AML)

Introduction: While AML is phenotypically diverse, genome-wide sequencing studies indicate that the median number of non-synonymous mutations is eight (Science 339,1546, 2013) and that the same pathways are affected in tumors with distinct genetic alterations (Nature 453,1112-1116, 2008). These insights provided the impetus to immunoaffinity purify shared antigenic moieties in human AML and examine their prevalence as well as function.Methods: We employed a previously described method wherein immunization of rabbits with normal human peripheral blood cells whose surface charge had been modified by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross reacted against a broad range of human leukemias (Nature 232,197, 1971). Replicating the described methods WBCs from healthy volunteer donors were suspended at 1x107 cells per ml of PBS and mixed with equal volume of 0.86 mM FDNB for 12 to 15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells (control rabbit). After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested in clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was performed using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immunoprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined by qRT-PCR and from raw data from Gene Expression Omnibus at the NCBI. IQGAP1 expression was evaluated by immunohistochemistry in cell lines, bone marrow biopsies from AML patients and normal healthy volunteers. IQGAP1 expression was modulated by RNAi and the effect on proliferation and colony formation was measured in cell lines.Results: Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immuno-precipitation of antigens from whole cell lysates of clinical AML samples revealed the scaffolding protein IQGAP1 as being differentially recognized in independent experiments. We focused on IQGAP1 because it regulates multiple cellular functions including signaling pathways involved in proliferation and transformation and stimulates cell motility and invasion. It is overexpressed in colon cancer, pancreatic cancer, hepatocellular carcinoma, glioma, and other cancers. (Cell Signaling 21,1471-1678, 2009). Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band, see figure. There was 3 to 9-fold greater expression of IQGAP1 expression in AML versus normal bone marrow observed by qRT-PCR. Analysis of raw data from Gene Expression Omnibus at the NCBI revealed fold change of -3.22636, p-value 2.62 x 10e-7 between normal bone marrow versus AML. Preliminary IHC studies showed strong membranous staining in three leukemia cell lines and bone marrow biopsies from primary AML bone marrow samples but weak partial staining in normal bone marrow. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation as compared to effects of scramble control. Mean and SD of colonies per 2000 cells plated were for K562: naïve 311+/-46, IQGAP1 knockdown 37+/-12, scramble control 127+/-12, MV4-11: naïve 156+/37, IQGAP1 knockdown 55+/- 8, scramble control 147+/-16, and THP1 naïve 232+/-45, IQGAP1 knockdown 17+/-2.9, scramble control 150+/-24.Conclusions: 1. The scaffolding protein IQGAP1is overexpressed in AML compared to normal bone marrow. 2. Knocking down expression of IQGAP1 in AML cell lines resulted in significant decrease in proliferation and colony formation providing proof of concept that IQGAP1 is a potential therapeutic target in AML. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Suppression of mTOR Signaling Attenuates Apoptotic Effects Induced By the NEDD8-Activating Enzyme Inhibitor Pevonedistat (MLN4924) in Acute Myeloid Leukemia Cells

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Therefore, novel therapies for AML are needed. Recently, the NEDD8 conjugation pathway and the mammalian target of rapamycin (mTOR) signaling pathway have emerged as important regulatory pathways for cancer therapy. Since some components of the mTORC1/2 pathways such as Deptor are neddylation targets, we postulated that combined exposure to NEDD8-activating enzyme (NAE) inhibitor pevonedistat and the dual mTORC1/2 inhibitor sapanisertib (TAK-228) might have additive or synergistic effects in AML.To test pevonedistat and sapanisertib effects on AML cells, we used the agents alone or in combination followed by assessment of proliferation, apoptosis and signal transduction in human AML cell lines as well as in primary cells. By AnnexinV/7AAD flow cytometry analysis, we found that pevonedistat significantly induced AML cell death and apoptosis in a time- and dose-dependent manner. Western blot showed that poly (ADP-ribose) polymerase (PARP) and caspases 3/8/9 were cleaved, indicating that pevonedistat-treated cells were actively undergoing apoptosis. Among the six AML cell lines (HL-60, KG1a, Molm-13, MV4-11, THP-1 and U937) tested, MV4-11 was the most sensitive cell line (IC50 <25nM) and KG1a was the least sensitive cell line (IC50 >1000nM) after 48 hours of exposure. The average IC50 on primary AML blast cells (5 primary samples) was 400nM at 48 hours. Western blot analysis showed that the neddylated cullin levels were markedly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. We also found that pevonedistat induced morphologic changes and increased CD11b expression in Molm-13 and MV4-11 cells, indicating cell differentiation.The effects of pevonedistat on AML cells could be attenuated by pre- or co-treatment with the mTORC1/2 inhibitor sapanisertib. We found that sapanisertib alone induced limited cell death in most AML cell lines and primary AML patient blasts. The results from XTT assay and flow cytometry analysis indicated that sapanisertib alone significantly decreased cell size, arrested cells in G0-G1 phase and reduced protein content of AML cells but did not induce robust apoptosis. This suggested that through inactivating mTOR signal pathway, sapanisertib suppressed cell activity and induced a dormant/quiescent status and thereby attenuated the apoptotic effects of pevonedistat. With concurrent pevonedistat and sapanisertib exposure, the expression of apoptotic mediators such as cleaved PARP and caspases 3 were decreased compare to pevonedistat treatment alone (See Figure).To confirm the role of mTOR inhibition on pevonedistat-induced AML apoptosis, we targeted mTORC1/2 by knocking down mTOR expression with small hairpin RNAs (shRNA). Downregulation of mTOR expression mirrored the effects of sapanisertib. mTOR inhibition by shRNAs resulted in decreased AML cell size and arrest of cells in G0-G1 phase. In addition, pevonedistat-induced cell death was significantly impeded in the cells infected with mTOR shRNA lentivirus compared to scrambled shRNA (p<0.01). Therefore, we validated that mTOR signal inactivation protects AML cells from NAE inhibitor pevonedistat-induced apoptosis using both pharmaceutical and genetic approaches. To further study the mechanism behind mTOR inhibition inducing resistance to pevonedistat, we tested rapamycin, an mTORC1 inhibitor. Concurrent exposure to rapamycin did not significantly alter the effects of pevonedistat and did not change cell size and cell cycle status. In addition, rapamycin did not inhibit the phosphorylation of 4E-BP1, suggesting that mTORC1 inhibition alone may be insufficient to inhibit the effects of pevonedistat, and suggesting a role for mTORC2.Taken together, our data demonstrate that the NEDDylation pathway inhibitor pevonedistat has strong potential as a therapeutic agent for AML therapy via apoptosis induction. However, when AML cells are arrested in G0-G1 with reduced proliferation rates by dual mTORC1/2-inhibition, the ability of pevonedistat to induce apoptosis is suppressed. Whether this is due solely to the cytostatic effects of dual mTOR inhibitors or also involves other mechanisms remains uncertain. While both sapanisertib and pevonedistat have clear anti-leukemia effects, their concurrent use may inhibit NAE inhibitor-induced apoptosis. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Inhibitory Effect of Histone Deacetylase Inhibitor on the Proliferation of Leukemia Cells and Its Anti-Tumor Pharmacology

The aim of this study was to explore the inhibitory effect of histone deacetylase inhibitor (HDACI) on the proliferation of leukemia cells. The two kinds of leukemia cells (human promyelocytic leukemia cell (HL-60) and human acute myelogenous leukemia cell (KG-1)) were selected for in vitro research. Besides, Chidamide, a kind of benzamide HDACI, was applied to induce and culture the HL-60 and KG-1 cells, and the anti-tumor cell proliferation activity of Chidamide on HL-60 and KG-1 was detected by the methyl thiazolyl tetrazolium (MTT) assay, which was 5.6 and 6.1 in turn. The cell scratch experiment verified that Chidamide had the metastasis inhibitory effect on HL-60 and KG-1 cells. Flow cytometry was employed to measure the percentage of apoptotic cells, and it was found that the percentage of apoptotic cells was 55.6% ± 1% and 48.6% ± 1% in sequence after HL-60 and KG-1 cells were treated with Chidamide for 36 hours. The number of auto-phagosomes was determined by transmission electron microscopy showing that the number of auto-phagosomes in HL-60 and KG-1 cells was 12 ± 1 and 10 ± 1, respectively after the induction process of Chidamide. The phosphorylated histone H2AX protein (γ-H2AX) recognition antibody immunofluorescence method was adopted to determine the deoxyribonucleic acid (DNA) damage, and the positive rates of HL-60 and KG-1 cells reached 28.41% and 26.35%, respectively after Chidamide treatment. Therefore, Chidamide, as a kind of HDACI, could effectively inhibit the proliferation of leukemia cells, so that the results of this experiment had a good guiding meaning for the clinical diagnosis and treatment of leukemia.

A novel dipeptide type inhibitor of the Wnt/β-catenin pathway suppresses proliferation of acute myelogenous leukemia cells

The Wnt/β-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/β-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated “31” that was found to be an inhibitor of the Wnt/β-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/β-catenin signaling pathway.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Abstract I11: Targeting pancreatic cancer with TCR-engineered T cells

Abstract We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T-cell responses by transfer of genetically engineered T cells. Our largest clinical experience has been in treating human acute myelogenous leukemia (AML), in which we have utilized a high-affinity TCR specific for WT1, a protein associated with promoting leukemic transformation that is overexpressed in human leukemic stem cells, to genetically engineer CD8 T cells. We recently reported a study (Chapuis et al., Nat Med 2019) in which we treated leukemia patients at high risk of relapse (after hematopoietic cell transplant) that demonstrated all treated patients remain alive and relapse free at a median of 48 months, compared to a relapse rate of ~35% in the concurrent matched cohort (p&amp;lt;0.01). We have also been developing strategies to translate insights and technologies from this study to treatment of solid tumors. In a preclinical genetically engineered mouse model (KPC mice) of pancreatic cancer that faithfully replicates most aspects of human disease, we demonstrated (Stromnes et al., Cancer Cell 2015) that CD8 T cells engineered with a high-affinity TCR specific for mesothelin (Msln) can infiltrate pancreatic tumors, mediate antitumor activity, and provide therapeutic benefit. However, since the T cells are ultimately rendered dysfunctional in the tumor microenvironment (TME), prolonging survival has required repeated infusions of T cells to sustain antitumor activity. We have now isolated and validated a human high-affinity TCR specific for Msln for use in a planned clinical trial modeled after the approach successful in KPC mice. However, we would like to both enhance and sustain antitumor activity without requiring repeated infusions. In-depth analyses of the T cells, tumors, and the TME in treated KPC mice have illuminated strategies to potentially overcome the obstacles to tumor eradication, and we have been exploring molecular engineering approaches to achieve this. One approach has been to create synthetic immunomodulatory fusion proteins (IFPs) that have an ectodomain composed of the receptor for an inhibitory ligand encountered in the TME but, rather than the natural cytoplasmic tail that would deliver an inhibitory signal, the receptor has the tail of a costimulatory receptor and delivers an activation signal. Expression of such IFPs takes advantage of the inhibitory ligands commonly encountered in the TME by T cells by co-opting potential inhibitory signals and has resulted in enhanced T cell function, persistence/survival, and antitumor activity. Another major obstacle to sustained therapeutic activity appears to be the limited access in the TME to nutrients that effector T cells can utilize as an energy source. Analysis of the metabolites present in the TME and the transcriptional program in T cells has provided insights into genetic modifications that can be made to allow T cells to survive and function in the metabolically hostile TME. These and related studies will be discussed. Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Luigi Nezi, Teresa Manzo, Shannon K. Oda, Kelly G. Paulson, Rachel Perret, Leah Schmidt, Tom M. Schmitt, Ingunn M. Stromnes, Aude G. Chapuis. Targeting pancreatic cancer with TCR-engineered T cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I11.

Acceleration of AML Progression By Cigarette Smoke Exposure or Condensate Exposure and Associated DNA Methylation Alterations

Subsets of acute myelogenous leukemia (AML) are characterized by molecular alterations with prognostic significance, however, little is known about how modifiable behaviors, such as cigarette smoking, intersect with genetic factors. Mutations rendering the Fms-like tyrosine kinase 3 (FLT3) constitutively active, such as internal tandem duplication (ITD), are associated with refractory disease and therapy resistance. Inhibition of the FLT3 kinase shows some benefit in this population, as highlighted by the FDA approvals of midostaurin and gilteritinib, but overall outcomes remain poor. Cigarette smoke exposure (CSE) also marks a population of patients with poor prognosis. Current and former smokers who develop AML are known to have worse survival as compared to never smokers (Alfayez M., et al. ASCO 2019), but the impact of FLT3 mutation and subsequent associated treatment response has not been studied. Also, the underlying mechanism of how history of cigarette smoking influences leukemia biology and response to therapy is poorly understood. In order to model a history of smoking in AML patients, NOD-SCID mice (n=25) were exposed to CSE using a smoking robot for 2 hours, 5 days/week, for 2 weeks. Mice were then inoculated via tail-vein injection with luciferase tagged human FLT3-ITD cells and leukemia burden was monitored through noninvasive imaging. CSE continued through the duration of the experiment, post engraftment. Control "non-smoking" mice (n=15) were only injected with leukemia cells. Within one week post leukemic introduction, a significant increase in leukemic burden as measured by bioluminescence was apparent in mice exposed to CSE versus control mice (P-value&lt;0.0001). To model the impact of smoking cessation upon diagnosis of AML in patients, experiments were modified to halt CSE once leukemic engraftment was detectable by non-invasive imaging. Smoking cessation versus continuous smoke exposure yielded reduced relative leukemic burden. Mice with continuous smoke exposure had higher rates of leukemia compared to mice who ceased smoking (n=10) one week prior (P-value =0.0064). These rapid changes in leukemic burden suggest that CSE may prime the microenvironment to promote leukemia progression or directly affect leukemia cells. To address the latter possibility, human AML cells were exposed to cigarette smoke condensate (CSC), which contains the chemicals present in cigarettes, for two weeks before introducing the cells into mice. A significant increase in leukemic burden was observed in mice injected cells exposed to CSC compared to mice injected with unexposed leukemia cells (P-value &lt;0.001), highlighting a direct role for the chemicals in cigarettes on in vivo leukemia proliferative factors. Smokers are known to carry altered global DNA methylation signatures that persist decades after quitting. To measure DNA methylation changes in the in vivo models described above, we examined spleens of non-smoking and smoke exposed mice by reduced representative bisulfite sequencing (RRBS). Sequences were mapped to either the human or mouse genome, (enabling identification of leukemia specific versus microenvironment specific alterations) and were compared in the smoking and non-smoking mice. Over 200 genes exhibited significant DNA methylation alterations in their promoter regions. Genes involved in RNA polymerase activity and chromatin remodeling were highly represented amongst those with altered DNA methylation. The clinical significance of our observations was confirmed in a cohort of 58 treatment naïve FLT3-ITD AML patients at MD Anderson receiving intensive induction therapy: 41 never smokers and 17 ever smokers. Smokers had significantly reduced survival as compared to the never smokers (median overall survival of 18 vs 23 months, P-value 0.0092). Collectively our findings indicate that short-term CSE is sufficient to alter DNA methylation patterns and accelerate the early progression of FLT3-ITD AML in vivo. Smoking cessation upon diagnosis may slow leukemic growth relative to smoking throughout AML therapy prompting the consideration of behavioral interventions for smokers with AML. Improved understanding of the mechanism of leukemic progression and drug resistance from CSE is expected to lead to improved treatment paradigms designed for patients with a history of cigarette smoking. Disclosures Konopleva: Genentech: Honoraria, Research Funding; Kisoji: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents &amp; Royalties; Ablynx: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. DiNardo:agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; jazz: Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria; syros: Honoraria; celgene: Consultancy, Honoraria.

Novel Benzo[B]Furans with Anti-Microtubule Activity Upregulate Expression of Apoptotic Genes and Arrest Leukemia Cells in G2/M Phase.

Novel derivatives of benzo[b]furan were found to be highly toxic towards human chronic myelogenous (K562), acute myelogenous (HL-60) and acute lymphoblastic (MOLT-4) leukemia cells. The objective was the characterization of the biological activity of novel benzofurans (influence on apoptosis, mitogen-activated protein kinases and on the cell cycle). Cellular protein(s) targeted by test benzofurans and mechanism of action were identified. The methods utilized in the study were chemical synthesis, fluorescence assays, flow cytometry, gene expression by DNA microarray and real-time RT-PCR, western blotting, cytotoxicity assays, pull-down assay, mass spectroscopy, in vitro polymerization of tubulin, molecular docking. 1,1'-[3-(bromomethyl)-5,6- dimethoxy-1-benzofuran-2,7-diyldiethanone (1) and methyl 4-bromo-6- (dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (2) induced apoptosis in K562 and MOLT-4 cells. The profiling of gene expression revealed that 1 and 2 increased the expression of proapoptotic genes involved in both receptor (TNFRSF 10A, TNFRSF 10B, CASP8) and mitochondrial (BAX, BID, NOXA, APAF1) pathways of apoptosis. Test benzo[b]furans activated c-Jun N-terminal kinase (JNK) and p38 kinase in K562 cells. Tubulin was identified as a protein target for benzo[b]furans in pull-down experiments with biotinylated 2. Test benzo[b]furans inhibited polymerization of tubulin monomers in vitro, decreased the level of cellular microtubules and arrested cells in a G2/M phase. Molecular docking suggests that benzo[b]furans 1 and 2 bind to tubulin via colchicine binding pocket and the complex is stabilized mainly by hydrophobic interactions. Novel benzo[b]furans with anti-microtubule activity were identified. They induce apoptosis in cancer cells and cause G2/M cell cycle arrest. Biological activity of 1 and 2 makes them potential lead compounds for development as anticancer drugs.

Pivotal role of mitophagy in response of acute myelogenous leukemia to a ceramide-tamoxifen-containing drug regimen

Acute myelogenous leukemia (AML) is a hematological malignancy marked by the accumulation of large numbers of immature myeloblasts in bone marrow. The overall prognosis in AML is poor; hence, there is a pressing need to improve treatment. Although the sphingolipid (SL) ceramide demonstrates known cancer suppressor properties, it’s mechanism of action is multifaceted. Our studies in leukemia and other cancers have demonstrated that when combined with the antiestrogen, tamoxifen, the apoptosis-inducting effect of ceramide is greatly enhanced. The goal of the present study was to establish whether a ceramide-tamoxifen regimen also affects autophagic-driven cellular responses in leukemia. Using the human AML cell line KG-1, we demonstrate that, unlike exposure to the single agents, combination C6-ceramide-tamoxifen upregulated LC3-II expression, inhibited the mTOR signaling pathway, and synergistically induced KG-1 cell death in an Atg5-dependent manner. In addition, colocalization of autophagosome and mitochondria, indicative of mitophagosome formation and mitophagy, was observed. Versatility of the drug regimen was confirmed by experiments in MV4-11 cells, a FLT3-ITD AML mutant. These results indicate that the C6-ceramide-tamoxifen regimen plays a pivotal role inducing autophagy in AML, and thus constitutes a novel therapeutic design.

Abstract IA17: Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors

Abstract We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. Our largest clinical experience is in treating human Acute Myelogenous Leukemia (AML). After identifying that WT1, a gene associated with promoting leukemic transformation, is over-expressed in human leukemic stem cells, and demonstrating in a clinical trial that in vitro expanded WT1-specific CD8 T cell clones can be safely transferred, exhibit anti-leukemic activity, and provide therapeutic benefit to AML patients, we extensively screened normal human repertoires and isolated a high affinity TCR specific for WT1 for genetically engineering CD8 T cells to reproducibly create cells with high avidity for leukemic cells. We have initially pursued this strategy in a 2 Arm trial for leukemia patients either at high risk of relapse (Arm 1) after hematopoietic cell transplant (HCT) or who have already relapsed after HCT (Arm 2). The prophylactic arm is now completed, with very encouraging results- all patients treated with engineered T cells remain alive and relapse free at a median of 38 months, compared to a relapse rate of ~50% in a concurrent matched cohort. Results in relapsed patients (Arm 2) have been less effective, and we have used high-dimensional analyses including single cell RNAseq both to elucidate the reasons for failure to eradicate the leukemia and to design strategies to overcome these obstacles. Our results have identified several mechanisms by which the leukemia escapes, and we have been testing approaches that employ further genetic modification of the T cells to enhance efficacy. The predominant reason for leukemia progression despite targeted therapy with T cells is the inability of the T cells to persist and maintain function in the context of encountering a rapidly proliferating myeloid leukemia. This reflects both engagement of pathways inhibitory to T cells by the leukemic cells, and apoptosis of the T cells from repetitive stimulation. We are addressing this issue by creating immuno-modulatory fusion proteins (IFPs) that have the ectodomain of an inhibitory or death receptor fused to a survival costimulatory domain. Results with two such IFPs will be discussed, a CD200R/CD28 fusion that binds the inhibitory ligand CD200 commonly expressed on leukemic cells but provides a CD28 costimulatory signal and a Fas/4-1BB fusion that binds FasL but rather than induce death promotes proliferation and survival. A more uncommon reason explaining progression is loss of expression of the WT1 epitope being targeted. This has occurred in 2 patients, but for distinct reasons. In one patient this reflected loss of a component of the immunoproteasome, and we have now isolated a TCR that recognizes an epitope not dependent on the immunoproteasome. In a second patient the level of WT1 expression declined- interestingly, this patient was treated with Vidaza, which can increase expression of WT1, and post-Vidaza the transferred T cells persisting in the patient’s bone marrow recognized and responded in vivo to the relapsing leukemia. The approaches and technologies we are developing and testing in leukemia are also applicable to solid tumors, and preclinical studies in pancreatic and ovarian cancers will be discussed. Citation Format: Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Kelly Paulson, Rachel Perret, Leah Schmidt, Thomas Schmitt, Ingunn Stromnes, Aude Chapuis, Philip D. Greenberg. Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA17.

The aberrant epigenetic regulation and epigenomic landscape alteration in human acute myelogenous leukemia and the emerged agents that target epigenetic regulators

The aberrant epigenetic regulation and epigenomic landscape alteration in human acute myelogenous leukemia and the emerged agents that target epigenetic regulators

Monitoring ESAM Expression Levels Reveals Autonomous Fluctuation of Leukemia Stem Cells By Autocrine/Paracrine Cytokine Signaling

Recently , we showed that hematopoietic stem cells (HSCs) fluctuate in a dynamic trajectory for self-renewal and lineage commitment, which regulates HSC heterogeneity (Cell Rep 2018). Although the significance of heterogeneity is difficult to interpret, it might enable HSCs to maintain a dynamic equilibrium of homeostatic hematopoiesis and to adapt promptly to stress-induced hematopoiesis based on physiological demands. We hypothesized that similar to HSCs, leukemia stem cells (LSCs) are heterogeneous with active fluctuation, which contributes to treatment resistance. Hence, this study aimed to show the fluctuation of LSCs and determine the underlying molecular mechanisms.We previously reported that the endothelial-cell selective adhesion molecule (ESAM) is useful as a life-long marker for HSCs in humans and mice (Blood 2009, Exp Hematol 2013). ESAM expression levels precisely indicated the activation status of HSCs (J Immunol 2012). Furthermore, approximately two-thirds of patients with acute myelogenous leukemia (AML) were positive for ESAM expression on leukemia cells in a heterogeneous manner. (Exp Hematol 2013). We exploited human AML lines as LSC-like cells and monitored expression levels of ESAM. Flow cytometry analyses revealed wide-ranging ESAM expression levels on KG1a and CMK lines. In KG1a cells, ESAM-high cells were concentrated in the CD34+CD38- fraction with high growth capability, whereas ESAM-low cells were less proliferative. Notably, when sorted into ESAM-high or ESAM-low subgroups, both groups reconstituted the parental heterogeneous distribution of ESAM levels in culture. Furthermore, single self-renewing LSC-like clones also reproduced the same heterogeneous population regardless of their original ESAM levels. These results suggested that LSC-like clones developed the heterogeneous AML cell population by fluctuating, which could be monitored with ESAM levels.The transcriptomes of ESAM-high and ESAM-low LSC-like cells in KG1a line were compared to obtain comprehensive information of key molecules involved in fluctuation and heterogeneity. We found that expression of T-cell immunoglobulin and mucin domain 3, reported as a reliable LSC marker for human AML (Cell Stem Cell 2010), was variable in parallel with ESAM. In addition, network analysis demonstrated that “genes related to hematopoietic progenitor cells” were activated whereas “genes related to blood cell maturation” were downregulated in ESAM-high cells. These results suggested that ESAM-high LSC-like cells were more identical to the conventional concept of LSCs than ESAM-low cells. However, expression levels of BAALC, SPP1, and ITGB7 were elevated in ESAM-low cells. BAALC encodes “brain and acute leukemia cytoplasmic (BAALC)” protein, which inhibits drug-induced apoptosis in KG1a cells (Hematology 2012). SPP1 encodes “secreted phosphoprotein 1 (SPP-1), also known as osteopontin, which induces chemo-resistance in AML cells (Life sci 2017). Furthermore, a recent paper reported that the active form of integrin □7 can be a therapeutic target of myeloma stem cells (Nat Med 2018). Thus, we infer that fluctuating to the ESAM-low stage may contribute to the refractory nature of LSCs in AML.We performed an up-streaming analysis on the transcriptome data to determine the signaling pathways involved in the fluctuation. The tumor necrosis factor alpha (TNFα) and the transforming growth factor beta (TGFβ) signaling pathways were found to affect ESAM expression levels. When TNFα or TGFβ1 was added to culture medium, the proliferation of KG1a cells was suppressed in a dose-dependent manner. Furthermore, addition of TNFα or TGFβ1 significantly suppressed ESAM expression levels on a subset of KG1a cells, which enhanced their heterogeneity. When cultured in serum-free cytokine-free medium, KG1a cells became uniform in terms of ESAM expression levels, with the ESAM-low population disappearing . Intriguingly, we found that KG1a cells produced TNFα and TGFβ1 autonomously, which induced the cell heterogeneity in an autocrine/paracrine manner. Surprisingly, blocking these signaling pathways by receptor inhibitors induced apoptosis in KG1a cells. These results suggest that autocrine/paracrine cytokine signaling plays important roles in promoting the variable and heterogeneous nature of LSCs, the inhibition of which might be a strategy for therapeutic intervention in refractory AML. DisclosuresYokota:Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Jansen Pharmaceutical K.K: Honoraria; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.