ALBERT

Description: PURPOSE: Recent progress of the treatment of multiple myeloma (MM) has significantly improved prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. The purpose of this study is to find compounds effective for high-risk myeloma with lower toxicity by developing chemical structure of the phthalimide. We also tried to isolate their binding molecules to understand molecular pharmacology of the compounds. RESULTS: (1) Screening and optimization of phthalimide derivatives: We screened library of synthetic phthalimide derivatives by their ability to induce apoptosis of MM cell line, KMS34, which has high-risk cytogenetic abnormalities such as t(4;14) and TP53 gene deletion. We finally found TC11, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione, which showed the most potent tumor growth inhibition. Based on the results of screening of phthalimide library, we found the two important points of chemical structure: (i) A 4-amino group lacked growth inhibitory effect, and thus, 5-amino branch is necessary for strong anti-tumor activity. (ii) Modification of 2,6-diisopropylphenyl in phenyl ring of TC11 significantly decreased inhibitory effects of tumor cell growth. (2) Anti-myeloma effects of phthalimide derivatives: TC11 showed significant growth inhibition of all myeloma cell lines (IC50=3-8μM) including those with high-risk cytogenetic changes. Lenalidomide also significantly inhibited growth of myeloma cell lines. However, 30μM of lenalidomide failed to inhibit growth of KMS34 and KMS28 cell lines that have high-risk cytogenetic alterations. TC11 also inhibited growth of bone marrow myeloma cells obtained from the MM patients. (3) Anti-myeloma effects of TC11 in vivo: Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous plasmacytoma in human myeloma cell (KMS34 and KMS11)-bearing SCID xenografts. In the pharmacokinetic analyses, the Cmax was 2.1μM at 1 h after the injection of TC11, with 1.2 h as the half-life. Cmax was 18.1μM at 1.5hr (Tmax), and T1/2 was 4.5hr, when 100mg/kg of TC11 was injected. Cmax was 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr, if 20mg/kg was injected. In oral administration of 200mg/kg of TC11 in lcr mice, Cmax was 3.81μM at 8.0hr (Tmax), and T1/2 was 2.77hr. (4) Toxicity of TC11 and lenalidomide: When 20mg/kg of TC11 was injected to SCID-mice for two weeks, weight loss compared with control mice was not observed. Clonological assay using mice bone marrow cells showed that 10μM TC11 did not suppress the colony formation while 5μM lenalidomide significantly decreased the colony out put. Therefore, TC11 in the concentrations that induced apoptosis of myeloma cells unlikely caused systemic and hematological toxicity. (5) Binding molecules: To understand the molecular mechanism, we employed our unique in vitro screening system using mRNA display, in vitro virus (IVV) method. It was found that TC11 directly bound to nucleophosmin 1 (NPM1) and α-tubulin. Knock-down of NPM1 gene in myeloma cells significantly delayed tumor cell growth. However, cereblon (CRBN), which was considered to be associated with teratogenicity of thalidomide, was not included in TC11-binding molecules in IVV assay. TC11 lacked glutarimide moiety to which CRBN was reported to bind. Thus, TC11 revealed anti-myeloma activity in a CRBN-independent pathway and is conceptionally expected as a non-teratogenic thalidomide-related compound. Interestingly, lenalidomide also directly bound to NPM1. Further analyses were needed to elucidate biological significance of interaction of IMiDs with NPM1 and α-tubulin. CONCLUSION: Our results suggested the possibility that phthalimide derivatives induce tumor cell death independent of CRBN pathway. It was also suggested that drug design and modification of chemical structure of phthalimide enable us to develop further new thalidomide derivatives which have more potent antitumor activity and less toxicity. Disclosures Ichikawa: Takeda Pharmaceutical Company: Research Funding. Matsushita:Takeda Pharmaceutical Company: Research Funding. Hattori:MSD company: Research Funding; Ono Pharmaceutical company: Research Funding; Takeda Pharmaceutical Company: Consultancy; Novo Nordisk company: Research Funding; Mitsubisi Tanabe Pharm: Research Funding; Cosmic Corporation: Research Funding.

Description: Background: Bortezomib (BTZ), a proteasome inhibitor (PI), mainly targets the beta 5 subunit of the 20S proteasome, and is widely used in the treatment of multiple myeloma (MM). However, inhibitory effects on other subunits of the proteasome are not well understood. Therefore, we examined the anti-MM activity of novel syringolin analogs that inhibit the activity of both beta 5 and 2 subunits. After examination, we investigated the activity of compound 19a, developed as a syrbactin-class PI to improve cytotoxic activity and membrane permeability (Chiba T et al. Angew Chem Int Ed. 2014). Materials and method: First, the cytotoxic and inhibitory effects of compound 19a on 20S proteasomes of MM cells, including BTZ-resistant MM cells and primary samples derived from MM patients, were examined. The primary MM specimens were collected after obtaining written informed consent at Nagoya City University Hospital, and the mechanism of antitumor activity of compound 19a on MM cells was evaluated by focusing on the unfolded protein response and endoplasmic reticulum (ER) stress. Finally, to evaluate the toxicity of compound 19a, BALB/c mice were intraperitoneally injected with either 19a or BTZ and body weight change was analyzed. These in vivo experiments were performed in accordance with the United Kingdom Coordinating Committee on Cancer Research Guidelines for the Welfare of Animals in Experimental Neoplasia, Second Edition, and were approved by the Ethics Committee of the Center for Experimental Animal Science, Nagoya City University Graduate School of Medical Sciences. Results: The cytotoxic activity of compound 19a, observed in various MM cell lines at nanomolar concentrations, was found to be similar to the activity observed when BTZ-resistant MM and T-cell lymphoma cell lines were tested. More precisely, the two BTZ-resistant cell lines KMS-11/BTZ and OPM-2/BTZ showed 44.4-fold and 52.1-fold higher resistance, respectively, to BTZ than that shown by their parental cell lines KMS-11 and OPM-2. However, the two BTZ-resistant cells showed only 3.2-fold (IC50: 18.0 nM) and 4.3-fold (IC50: 5.1 nM) higher resistance to compound 19a than that shown by their parental cell lines KMS-11 (IC50: 5.7 nM) and OPM-2 (IC50: 1.2 nM), suggesting that compound 19a exhibits less cross-resistance to BTZ. Evaluation of 20S proteasome activity showed time-dependent inhibition of both beta 5 and 2 subunits in MM cells on treatment with compound 19a. Treatment with 10 nM 19a induced remarkable apoptosis of the MM cells, accompanied by elevated CHOP and NOXA expression, indicating excessive ER stress. A similar activity was also observed in primary MM samples derived from the patients. Furthermore, to clarify the effect of beta 2 inhibition on anti-MM activity, two MM cell lines, U266 and AMO1, and one T-cell lymphoma cell line, Hut78, were transfected with either siRNA-targeting PSMB7 encoding beta 2 subunit or control siRNA and were subjected to the analysis of cell growth and viability. Specific knocking down of PSMB7 was observed, which resulted in the progression of apoptosis of the two MM cell lines and Hut78 when compared with the control. In addition, knocking down of both PSMB7 and PSMB5 encoding beta 5 subunits triggered more potent apoptosis of U266 and Hut78 cells when compared with the knocking down of either PSMB7 or PSMB5 alone. This result suggests that dual-inhibitory activities of beta 5 and 2 subunits have an additive or a synergistic effect on cytotoxicity when compared with single inhibitory activity. Finally, to examine the toxicity of compound 19a, BALB/c mice were administrated with it in a dose-escalation manner and subjected to the analysis of alteration of body weight. No significant difference in loss of body weight was observed between 19a-treated and BTZ-treated mice when administered with the same dose. Study of in vivo cytotoxic activity of 19a on xenografted MM cells is currently underway. Conclusion: We demonstrated the cytotoxic activity of a syringolin analog on various MM cells at nanomolar levels, which was attributed to the dual inhibition of beta 5 and 2 subunits of the 20S proteasome. Compound 19a, as a dual inhibitor of beta 2 and 5, was observed to be a more potent PI than BTZ, and could overcome the acquired BTZ resistance. The findings provide new insight into the treatment of relapse and/or refractory MM. Table Table. Disclosures Ishida: Celgene KK: Research Funding; Kyowa Hakko Kirin, Co., Ltd.: Honoraria, Research Funding; Bayer Pharma AG: Research Funding. Iida:Janssen Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Description: Introduction Paraneoplastic pemphigus (PNP), an autoimmune disorder that can arise from malignancies, is fatal when complicated with bronchiolitis obliterans (BO), similar to that in patients receiving hematopoietic stem cell transplantation (HSCT). Autoantibodies to the desmosome proteins envoplakin and periplakin of epithelial cell adhesion molecules are essential for diagnosing PNP. BO after HSCT is an unfavorable complication that develops in association with chronic graft-versus-host disease (cGVHD). The effectiveness of rituximab on BO has been reported, which suggests that BO after HSCT is associated with autoimmunity similar to PNP. Here we analyzed anti-envoplakin and anti-periplakin antibodies that are associated with PNP in patients with and without BO complications who developed cGVHD after HSCT. Patients and Methods We analyzed anti-envoplakin and anti-periplakin antibodies in 21 patients (11 males and 10 females) with (n = 10) or without (n = 11) BO complications who developed cGVHD following HSCT between 1988 and 2015 at our institution. The median age at the time of HSCT in patients with and without BO was 10.8 (range, 1-21) and 7.5 (range, 1-14) years, respectively. The median duration from HSCT to BO onset was 261.6 (range, 41-825) days. We analyzed anti-envoplakin and anti-periplakin antibodies in cryopreserved sera collected before and after BO or cGVHD onset using immunoprecipitation (IPP) and enzyme-linked immunosorbent assay (ELISA) for detecting both autoantibodies. Longitudinal sera were obtained from a patient with cGVHD. Fifty-eight serum samples were collected from 21 patients. Biotinylated recombinant periplakin and envoplakin were produced from cDNAs using the transcription and translation (TnT) T7 Quick Coupled Transcription/Translation System (Promega) with rabbit reticulocyte lysates. IPP and ELISA were performed using in vitro TnT products as previously reported (Muro Y, et al. Rheumatology 2012;51:1508-13). Results On SDS-polyacrylamide gel electrophoresis, biotinylated recombinant periplakin and envoplakin showed protein bands with predicted size of 190 kDa and 210 kDa, respectively. The serum samples from the PNP patient and normal control serum were examined in an ELISA titration study. These titration curves showed the possibility of quantitatively measuring these autoantibodies. Both anti-envoplakin and anti-periplakin antibodies were positive in two of 10 patients with BO at BO onset but negative in all 11 patients without BO during cGVHD. In one of the two patients with plakin family autoantibodies, titers of anti-periplakin and anti-envoplakin antibodies gradually decreased after administering rituximab five times and repeating plasmapheresis seven times and subsequently decreased to below cut-off level. Although oxygenation substantially decreased consistent with the clearance of autoantibodies, the patient died of invasive pulmonary aspergillosis during steroid tapering. In the other patient, plakin family autoantibodies in the sera became negative after administering rituximab nine times. The patient is alive, and the spirometry findings were normal 6 years after the clearance of both autoantibodies. Conclusions Our findings indicate that plakin family autoantibodies may participate in BO onset in some patients receiving HSCT. The detection of these autoantibodies in patients with BO may be a good biomarker of cGVHD, providing a rationale and target for repeated rituximab therapy. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children. Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with 〉0.005% PNH granulocytes or 〉0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA. Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH. Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Description: [Introduction] Recent development of novel drugs significantly improved prognosis of Multiple Myeloma (MM). Immunomodulatory drugs (IMiDs) and proteasome inhibitors play central roles in MM therapy. However expanded usage of lenalidomide (Len) has increased the number of Len-resistant patients. And limited information is obtainable with regand to Len-resistant mechanism, such as overexpression and genetic mutation of IMiDs-binding protein, celeblon (CRBN); thus, elucidating the Len-resistant mechanism and development of drugs overcoming the Len-resistance are very important for improving the outcome of MM. The purposes of this study are [1] to clarify the molecular mechanism of Len-resistance using Len-resistant MM cell lines [2] to confirm the mechanism of CRBN independent myeloma cell death by novel phthalimide-derivatives, TC11 and PEG(E)-TC11. [Method] [1] In our laboratory, Len-resistant cell lines, KMS21R, KMS27R and MUM24R have been established by long-term co-culture with low-dose Len. Using these cell lines, we examined expression of CRBN and the downstream molecules, IKZF1/3, IRF4 and c-MYC by western blotting. We also examined the mutation of CRBN in KMS27R. [2] We have originally developed a novel phthalimide-derivative, TC11 and PEG(E)-TC11 synthesized for improving water solubility. We examined whether TC11 and PEG(E)-TC11 induced cell death to Len-resistance MM or not. [Result] [1] First, we validated expression of CRBN and the down-stream molecules, which mediate pharmacological action of Len. Decreased expression of CRBN and subsequent up-regulation of down-stream IKZF1 were confirmed in KMS21R cell. In KMS27R cell, IKZF 1/3 expressions are increased without alteration of CRBN expression level. Thus, genetic mutation in CRBN or IKZF1 is suspected in KMS27R cells. In MUM24R cell, no significant change in the expression levels of the CRBN pathway molecules was confirmed, suggesting other molecular alternation than CRBN pathway. [2] TC11 significantly induced apoptosis of Len-resistant cells. We have previously reported that TC11 didn't bind to CRBN and TC11 directly bound to nucleophosmin1 (NPM1) and α-tubulin. It was found that TC11 induced G2/M arrest and subsequent apoptosis by inhibition of tubulin polymerization and NPM1 oligomerization. Fluorescence microscopy observation showed that TC11 treatment induced hyper duplication of centrosomes in MM cells. Water solubility and blood absorption of PEG(E)-TC11 were significantly improved compared with those of TC11. As a consequence, PEG-(E)TC11 significantly delayed tumor growth in xenograft model mice. [Discussion & Conclusion] [1] Our present data suggested diversity of Len-resistant mechanism in MM patients. For example, in KMS21R, decreased expression of CRBN was likely the cause of Len-resistance. In KMS27R, genetic mutation in CRBN-IKZF1 pathway caused inhibition of IKZF1 degradation. In MUM24R, the Len-resistant mechanism didn't relate to the CRBN pathway but to unknown molecular mechanism. Len-resistant cell lines are useful tools for studying Len-resistant mechanisms and developing drugs overcoming Len-resistance. [2]TC11 abrogated tubulin polymerization and NPM1 oligomerization, induced centrosome disruption and G2/M arrest. Since G2/M check point doesn't closely rely on p53. TC11 was able to induce apoptosis of MM cells with high-risk cytogenetic mutations such as deletion of TP53 gene. TC11 and PEG-(E)TC11 are expected as a candidate compound overcoming Len-resistance and high-risk MM. Disclosures Matsushita: Amgen: Research Funding. Hattori:Takeda: Research Funding; IDAC inc.: Research Funding.

Description: Background: Recent progress in the treatment of multiple myeloma (MM) has improved its prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. We have been focusing on komaroviquinone (KQN) which was isolated from Dracocephalum komarovi and had anti-tripanosomal activity. Since a number of anti-cancer drugs have been developed from natural products with anti-protozoal activities, we tried to examine anti-tumor effect of novel KQN-derivatives against high-risk MM cells. Results and Discussion: (1) Screening of KQN derivatives and the anti-myeloma activity: We screened total of 16 synthetic KQN-derivatives with regard to their ability to inhibit growth of MUM24 cells, established from a thalidomide resistant patient, harboring high-risk cytogenetic abnormalities such as t (4;14) and TP53 gene deletion. Two KQN-derivatives, GTN024 and GTN005 were found to inhibit proliferation of MUM24 (IC50: 3.29 µM, 16.25 µM, respectively). When MUM24 cells were co-cultured with 5µM GTN024 and 20µM GTN005, annexin-V-positive fraction significantly increased, suggesting that GTN024 and GTN005 induced apoptosis. (2) ROS production and anti-myeloma activity: Because GTN024 includes benzoquinone structure, we examined production of reactive oxygen species (ROS) by KQN-derivatives in MM cells. We found that GTN024 produced ROS and inhibited growth of MM cells, which was abrogated by pre-incubation with antioxidants. GTN005 does not have benzoquinone structure, and produced small amount of ROS. Its growth inhibitory activity did not change despite incubation of MUM24 cells with antioxidants. These results suggested that KQN-derivatives showed growth inhibition of MM cells via ROS production in GTN024 and ROS-independent mechanism in GTN005. (3) Toxicity of GTN024 and GTN005: To evaluate the effect of GTNs on normal hematopoiesis, we conducted colony formation assays using murine bone marrow cells. GTN024 significantly suppressed the production of total colonies of normal hematopoietic cells in 1µM or higher dose, but GTN005 did not inhibit the production of colonies in IC50 or higher dose. Therefore, GTN005 was not considered to be toxic to normal hematopoietic stem cells. (4) Structural optimization and anti-myeloma activity: To improve the ROS-independent anti-MM effect of GTN005, we modified the benzene ring which is a common structure of KQN-derivatives. We developed two new derivatives, GTN054 and GTN057, having 1,4-hydroxy benzene or 1-hydroxy-4-acetoxy benzene, respectively. These compounds significantly inhibited proliferation of MUM24 (IC50〈 7µM). The results suggested that hydroxyl benzene structure was necessary for potent anti-MM effect. These two derivatives induced ROS production in MUM24 cells. However, growth inhibition did not change by pre-incubation with antioxidants. Therefore, anti-MM effects of GTN054 and GNT057 were independent of ROS production. (5)Anti-tumor effects in vivo: Intraperitoneal injections of GTN024 (10mg/kg BW) significantly delayed the growth of subcutaneous KMS11-derived plasmacytoma in SCID xenografts. When GTN024 was injected to mice in 20mg/kg BW or lower dose, no significant organ damages or hematological toxicity was observed. In higher dose, 40mg/kg BW, hematological toxicity was not observed, but gastrointestinal obstruction occurred. By pharmacokinetic analysis of GTN024, the Cmax was 392nM at 15min after injection of GTN024 and the half-life was 23min when 20mg/kg BW was injected. Conclusion: Our results implicated the possibility of KQN-derivatives as drugs for overcoming high-risk MM. Disclosures No relevant conflicts of interest to declare.

Description: Background Azacitidine (AZA) is one of the hypomethylating agents (HMAs) proven to significantly prolong overall survival (OS) for patients with higher-risk myelodysplastic syndromes (h-MDS) via a prospective phase 3 trial. However, the optimal treatment schedule of AZA for h-MDS has not yet been prospectively determined. As a 7-day treatment schedule includes weekends, a 5-day administration has also been applied with h-MDS, though solid evidence has not been established. Based on this background, we planned to compare AZA treatment on 7-day and 5-day schedules. Patients Patients diagnosed with de novo or treatment-related MDS (FAB-defined RAEB and RAEB-t), aged 16 years or older were eligible for this study, if they showed adequate performance status (ECOG PS 0-2) and no history of HMA treatment or chemotherapy. Candidates for allogeneic stem-cell transplantation were excluded. Study design This was a multicenter, randomized, open-label, phase 3 trial to compare the efficacy of AZA treatment for 7-days (AZA-7) to 5-days (AZA-5). The primary endpoint was 2-year OS rate (2y OS). AZA was given subcutaneously at 75 mg/m² every 28 days. To test the non-inferiority of AZA-5 to AZA-7, 2y OS of AZA-7 was estimated at 30%, and the delta was defined as 11%, for which 410 patients were needed to complete the trial. However, because of the poor recruitment, this study closed prematurely, and the protocol-planned interim analysis (when 200 patients were registered) was performed. This protocol was approved by the IRB at each trial center. All patients provided written informed consent, and the trial was conducted in accordance with the Declaration of Helsinki. The efficacy of AZA was evaluated using IWG2006 criteria, and treatment was continued until study completion, or relapse, unacceptable toxicity, or disease progression was observed. Efficacy was analyzed by intention-to-treat. Secondary endpoints were the hematological response rate, 2-year leukemia-free survival (2y LFS) rate, the cytogenetic response, and occurrence of adverse events. Results Between January 2013 and Jun 2018, 201 patients were randomly assigned to AZA-7 (n=99) or AZA-5 group (n=102). The median age of all patients at the enrollment was 73.5 years old (range, 48 to 91). Between the two groups, there was no significant difference in the baseline characteristics, such as sex, type of MDS (de novo or treatment-related), percent of BM blasts, FAB and WHO classification, and IPSS and IPSS-R risk. After excluding 5 and 6 patients from AZA-7 and AZA-5, respectively (poor data entry, change in diagnosis), at the time of last follow-up, 59 and 67 patients died in AZA-7 and AZA-5, respectively. The Kaplan-Meier estimates of the 2y OS and 2y LFS were 35.1% (95% CI, 23.8 to 46.7) and 27.3% (95% CI, 17.3 to 38.3) for AZA-7, and 22.4% (95% CI, 13.1 to 33.3) and 20.5% (95% CI, 11.6 to 31.2) for AZA-5, respectively. There was no significant difference in the frequency of erythroid, platelet or neutrophil improvement, or hematological response (complete or partial response, or any hematological improvement) between the two groups. The most common non-hematological adverse event of grade 3 to 4 (in ≥20% of the patients in either group) was febrile neutropenia (33.7% for AZA-7, and 30.5% for AZA-5). Conclusion and Discussion Because of the premature termination of this trial, statistical analysis for the primary endpoint (2y OS of 35.1% for AZA-7, and 22.4% for AZA-5) could not provide any solid evidence. However, considering the difference of more than 10% in 2y OS, and no major differences in the safety profiles, we favor AZA-7 to AZA-5 for MDS patients with RAEB and RAEB-t. Figure Disclosures Kiguchi: Tejin Co., Ltd.: Research Funding; Taiho Pharmaceutical Co., Ltd.: Research Funding; SymBio Pharmaceutical Co., Ltd.: Research Funding; Celgene Co., Ltd.: Research Funding; Janssen Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharmaceutical Co., Ltd.: Research Funding; MSD CO., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myeres Squibb Co., Ltd.: Research Funding; Sanofi K.K., Ltd.: Research Funding; Celltrion, Inc.: Research Funding. Usuki:Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc: Research Funding, Speakers Bureau. Suzuki:kyowa Hakko Kirin: Consultancy, Honoraria; MSD k.k.: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria; takeda: Research Funding; Incyte and Pfizer: Research Funding; astellas: Research Funding; Celgene: Honoraria; Chugai: Honoraria, Research Funding; daiichi sankyo: Research Funding; eisai: Research Funding. Tomita:Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Taiho Pharma: Research Funding. Handa:Ono: Research Funding. Maeda:Janssen Pharmaceutical K.K..: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Iyama:Otsuka Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Factory: Honoraria; Astellas Pharma: Honoraria; Daiichi Sankyo: Honoraria; Allexion Pharma: Honoraria; CSL Behring: Honoraria. Yamauchi:Astellas, AbbVie: Consultancy; Pfizer, Chugai, Teijin, Solasia: Research Funding. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Japan Inc.: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding; FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria.

Description: Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated 〉2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p 〈 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Description: Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, 〈 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P