Abstract: The level of expression of the transcription factor PU.1 is a critical determinant of lineage commitment in normal hematopoiesis, and dysregulation of PU.1 leads to development of leukemia. In mice with targeted disruption of the PU.1 upstream regulatory element (URE), expression of PU.1 is decreased to 20% of wild type levels and results in development of acute myeloid leukemia (AML). These data suggests that tightly regulated PU.1 expression is important to maintain normal hematopoiesis and prevent leukemogenesis. Previously, we reported that AML1 (RUNX1) regulated PU.1 expression. Here we demonstrate that AMLl regulates PU.1 through 3 AML1 binding sites in the URE. Mice with targeted mutations in the 3 AML1 binding sites have decreased PU.1 expression in multiple hematopoietic lineages at multiple different developmental stages. Conditional targeting of AML1 in transgenic mice in which the URE homology region 2 (H2, containing all 3 AML1 binding sites) is used to drive expression of a reporter decreased reporter gene expression, suggesting that AML1 regulates PU.1 through these 3 sites in URE homology region 2. Using a second mouse model with a targeted mutation in the PU.1 binding site in the PU.1 URE (which is flanked by the 3 AML1 sites), we demonstrated that PU.1 indeed autoregulates itself through the URE. These results demonstrated that AML1 regulates PU.1 through the 3 AML1 sites in the URE. However, while low levels of PU.1 lead to leukemia, we have not observed frank leukemia development in AML1 conditional knockouts or in mice with targeted disruption of the 3 AML1 sites in the PU.1 URE. We hypothesized that this might be the case because disruption of AML1 or the AML1 sites reduces PU.1 levels to about 40% of wild type, but not as great as that found in PU.1 URE knockouts, which do progress to AML (20% of wild type). We hypothesized that downregulation of PU.1 as a result of binding of AML1/ETO fusion proteins to the URE might result in further reductions of PU.1 expression, and contribute to leukemogenesis. Therefore, we predicted that development of leukemia might be delayed in mice with mutations in the PU.1 URE AML1 DNA binding sites, and this was indeed the case in a modle using a retrovirus expressing the AML1/ETO9a form. We further explored the effect of AML1 and PU.1 binding on chromatin strucutre using chromatin immunoprecipitation (Chip) in the AML1 and PU.1 site URE knockin models, and found that AML1 is involved in H3K4me3 and H3/H4 acetylation of histone tails in the PU.1 URE, while PU.1 is involved in H3/H4 acetylation but not H3K4me3; H3K4 methylation and H3 acetylation decreased in AML1 sites mutant knockin mice and H3 acetylation decreased in PU.1 site mutant knockin mice. Mutation of the AML1 site in mice not only altered the chromatin structure of the URE region, but also interefered with the physical interaction between the URE and PU.1 promoter, as assessed by chromosome capture configuration (3C) assays. Interestingly, the AML1/ETO9a fusion oncogene has a unique role on the epigenetic status of the PU.1 URE in addition to its dominant effect on the 3 AML1 sites. AML1-ETO9 blocks the autoregulation of PU.1 through the PU.1 site in the URE. In summary, our data suggests that AML1 regulates PU.1 expression through 3 AML1 binding sites in the PU.1 URE by modifying chromatin structure in the URE region. In addition, PU.1 can autoregulate itself by facilitating similar epigenetic changes. Dysregulation of the epigenetic status by chromosome translocation products such as AML1-ETO might play an important role in leukemogenesis. First two authors contribute equally to this work.

Abstract: Background Crovalimab is a novel anti-human complement component 5 (C5) antibody currently under investigation as a therapy for paroxysmal nocturnal hemoglobinuria (PNH), a life-threatening disorder characterized by hemolytic anemia and thrombosis. In the Phase I/II COMPOSER trial (NCT03157635; Röth et al. Blood. 2020), crovalimab showed promise as a therapy for PNH in patients with or without prior treatment with C5 inhibitors. Eculizumab and ravulizumab are C5 inhibitors currently approved for the treatment of patients with PNH, yet treatment limitations remain. Some patients experience breakthrough hemolysis due to unsustained C5 inhibition, there may be a lack of efficacy in patients with C5 mutational variants, and the requirement for regular intravenous (IV) infusions contributes to the treatment burden. Crovalimab is engineered to have a significantly extended half-life, enabling subcutaneous (SC) administration once every 4 weeks (Q4W), which could significantly reduce the treatment burden on patients with PNH. Study Design and Methods The Phase III, randomized, open-label, active-controlled, multicenter COMMODORE 1 study (NCT04432584) is evaluating the efficacy and safety of crovalimab compared with eculizumab in adult and adolescent patients with PNH currently treated with complement inhibitors. The study design is divided into 2 parts: randomized arms (Arms A and B) for primary and secondary efficacy analyses and a descriptive arm (Arm C) for exploratory analysis in patients of clinical relevance and importance (Figure). Adult patients with PNH (aged ≥ 18 years) are randomized 1:1 to receive either crovalimab (Arm A) or eculizumab (Arm B). The crovalimab regimen includes a loading series of an IV dose on Day 1 followed by weekly SC doses for 4 weeks starting on Day 2. This is followed by SC Q4W maintenance dosing starting at Week 5. Arm B patients receive IV maintenance dosing starting on Day 1 and then once every 2 weeks (Q2W) for 24 weeks. The descriptive analysis arm (Arm C) patients will receive crovalimab (same dosing regimen as Arm A patients). It will include: 1. Adolescent patients (aged 12-17 years) currently treated with eculizumab 2. Patients (aged ≥ 12 years) currently treated with ravulizumab 3. Patients (aged ≥ 12 years) currently treated with eculizumab at a dose & gt; 900 mg and/or more frequent than Q2W 4. Patients (aged ≥ 12 years) with a known C5 polymorphism whose hemolysis was poorly controlled After 24 weeks of treatment, patients from each treatment arm can continue crovalimab or switch from eculizumab to crovalimab if their physician determines this is in their best interest. The primary efficacy objective for the randomized arms is to determine the noninferiority of crovalimab compared with eculizumab based on mean percent change in lactate dehydrogenase (LDH) levels from baseline to after 24 weeks of treatment. Secondary efficacy objectives are to determine the noninferiority of crovalimab compared with eculizumab based on (1) proportion of patients who achieve transfusion avoidance, (2) proportion of patients who experience breakthrough hemolysis, (3) proportion of patients who achieve stabilization of hemoglobin, and (4) mean change in fatigue as assessed by the Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire. The safety objective is to evaluate the safety and tolerability of crovalimab compared with eculizumab based on the incidence and severity of adverse events, including infections (meningococcal meningitis and other infections), injection-site reactions, infusion-related reactions, hypersensitivity, adverse events leading to study drug discontinuation, and type 3 hypersensitivity reactions in patients who switch to crovalimab from eculizumab or ravulizumab. Pharmacokinetic, immunogenicity, biomarker, and health status utility objectives will also be assessed. Disclosures Risitano: Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; RA pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees. Röth:Biocryst: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Apellis: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding. Kulasekararaj:Alexion Pharmaceuticals Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pu:F. Hoffmann-La Roche Ltd: Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; Pennsylvania State University: Patents & Royalties; SUNY Upstate Medical University: Current Employment. Nishimura:Chugai: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Wright:Genentech, Inc: Current Employment; F. Hoffmann-La Roche Ltd: Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Appius:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Sreckovic:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Vignal:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Munir:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; Alexion: Honoraria.

Abstract: Background Crovalimab is a novel anti-complement C5 antibody currently being studied as a treatment for paroxysmal nocturnal hemoglobinuria (PNH), a life-threatening disease associated with hemolytic anemia and thrombosis. Treatment with approved C5 inhibitors eculizumab or ravulizumab is effective, but can be limited by breakthrough hemolysis due to unsustained C5 inhibition, inadequate efficacy in patients with C5 mutational variants, and the requirement of regular intravenous infusions. Crovalimab is unique in that its properties allow for subcutaneous injections once every 4 weeks (Q4W) that can be self-administered. Additionally, crovalimab binds to C5 mutational variants. Promising results were obtained in the Phase I/II COMPOSER trial (NCT03157635; Röth et al, Blood. 2020) conducted in patients with PNH, with or without prior anti-C5 treatment. The efficacy and safety of crovalimab vs eculizumab will be evaluated in two Phase III, randomized, open-label trials in patients with PNH, with or without current complement C5 inhibition. Study Design and Methods COMMODORE 1 (NCT04432584) will enroll patients who are currently receiving complement C5 inhibitor therapy. This trial is divided into two parts (Figure). Patients aged ≥ 18 years will be randomized 1:1 to receive either crovalimab (Arm A) or eculizumab (Arm B) and will contribute to the primary efficacy analysis. Patients aged & lt; 18 years can be enrolled in an exploratory descriptive arm (Arm C). Arm A and C patients will receive crovalimab loading and subsequent subcutaneous Q4W maintenance dosing from Week 5. Arm B patients will receive eculizumab intravenous maintenance dosing from Day 1, Q2W for a total of 24 weeks. Patients in Arm A and Arm C can continue to receive crovalimab, and patients in Arm B can switch to crovalimab after 24 weeks of treatment, as determined by the treating physician. The primary efficacy objective is to determine the non-inferiority of crovalimab vs eculizumab based on percentage change in lactate dehydrogenase levels from baseline, averaged over weeks 21, 23, and 25. Secondary efficacy objectives are to determine the proportion of patients who experience breakthrough hemolysis, achieve transfusion avoidance or hemoglobin stabilization, as well as determine mean change in fatigue according to the Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire from baseline to Week 25. Safety and tolerability of crovalimab vs eculizumab will also be evaluated along with pharmacokinetic, immunogenicity, biomarker, and health status utility objectives. COMMODORE 2 (NCT04434092) will enroll patients not currently treated with C5 complement inhibitors. This trial is also divided into two parts (Figure). Patients aged ≥ 18 years will be randomized 2:1 to receive either crovalimab (Arm A) or eculizumab (Arm B) and will contribute to the primary efficacy analysis. Patients aged & lt; 18 years will be enrolled in an exploratory descriptive arm (Arm C). Arm A and C patients will receive crovalimab loading and subsequent subcutaneous Q4W maintenance dosing from Week 5. Arm B patients will receive induction doses of eculizumab intravenously QW for 4 weeks followed by maintenance dosing Q2W up to 24 weeks. Patients in Arm A and Arm C can continue to receive crovalimab, and patients in Arm B can switch to crovalimab, after 24 weeks of treatment, as determined by the treating physician. The primary efficacy objective is to determine the non-inferiority of crovalimab vs eculizumab, based on the proportion of patients who 1) achieve transfusion avoidance from baseline to Week 25 and 2) with hemolysis control from Week 5-25 (co-primary efficacy endpoints). Safety and tolerability of crovalimab vs eculizumab will also be evaluated along with pharmacokinetic, immunogenicity, biomarker, and health status utility objectives. Figure 1 Figure 1. Disclosures Kulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Akari: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Risitano: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Research Funding, Speakers Bureau; Samsung: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; RA Pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Achillion: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees; Jazz: Other: Lecture fees, Speakers Bureau; F. Hoffmann-La Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Lecture fees, Speakers Bureau; Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Speakers Bureau. Roeth: Novartis: Consultancy, Honoraria; Bioverativ, a Sanofi company: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Apellis Pharmaceuticals: Consultancy, Honoraria; Kira: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria. He: LongBio Pharma: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy. Pu: University of Arizona: Current Employment; Pennsylvania State University: Patents & Royalties; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees. Wright: Genentech, Inc.: Current Employment. Appius: F. Hoffmann-La Roche Ltd.: Current Employment, Current equity holder in publicly-traded company. Sostelly: F. Hoffmann-La Roche Ltd: Current Employment. Sreckovic: F. Hoffmann-La Roche Ltd.: Current Employment. Stanzel: F. Hoffmann-La Roche Ltd.: Current Employment, Current equity holder in publicly-traded company. Munir: F. Hoffmann-La Roche: Consultancy; Alexion: Honoraria. Nishimura: Apellis: Consultancy; Novartis: Consultancy; Chugai: Consultancy; Sanofi: Consultancy; Alexion: Consultancy; Roche: Consultancy; Biocryst: Consultancy.

Abstract: Besides their role as antigen presenting cells, human peripheral blood mononuclear cell and CD34+ cell-derived dendritic cells (DCs), now have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumor-cell killing activity by human cord blood cell-derived dendritic cells (CBDCs). We report here that human cord blood monocyte-derived DCs aquire the ability to kill tumor cells after activation with lipopolysaccharide (LPS) or interferon-γ(IFN-γ), associated with the enhanced TNF-α-related apoptosis-inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14-positive cells collected from cord blood from healthy volunteers were cultured with interleukin-4 and granulocyte-machrophage colony- stimulating factor for seven days to induce CBDCs, which showed no cytotoxicity. However, after activation with IFN-γ for additional 12 hours, CBDCs exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS for 12 hours induced cytotoxicity against Daudi and Jurkat cells as we detected in human peripheral blood monocytes- drived DCs (PBDCs). IFN-γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface Fas Ligand as analyzed by flow cytometry. Our results suggest that activated CBDCs can serve as immunological effectors against tumor cells, through TRAIL- dependent and Fas-independent mechanisms.

Abstract: Background Crovalimab is a novel anti-human complement component 5 (C5) antibody engineered to significantly extend half-life and enable subcutaneous (SC) administration once every 4 weeks in C5-mediated diseases. Based on the promising results of the Phase I/II COMPOSER trial (NCT03157635; Röth et al. Blood. 2020), crovalimab is currently under investigation as a potential therapy for paroxysmal nocturnal hemoglobinuria (PNH), a life-threatening disorder characterized by hemolytic anemia and thrombosis. Eculizumab and ravulizumab are C5 inhibitors currently approved for the treatment of patients with PNH, yet treatment limitations include breakthrough hemolysis due to unsustained C5 inhibition, lack of efficacy in patients with C5 mutational variants, and the treatment burden of regular intravenous (IV) infusions. Study Design and Methods The Phase III, randomized, open-label, active-controlled, multicenter COMMODORE 2 study (NCT04434092) is evaluating the efficacy and safety of crovalimab compared with eculizumab in patients aged ≥ 12 years with PNH not previously treated with complement inhibitors. Patients are randomized 2:1 to receive crovalimab or eculizumab (Figure 1). Two hundred patients in the crovalimab arm will receive a loading series of crovalimab (IV dose on Day 1, followed by weekly SC doses for 4 weeks starting on Day 2). This is followed by SC maintenance dosing every 4 weeks starting at Week 5. Patients in the eculizumab arm receive a weekly IV loading dose of eculizumab for the first 4 weeks, followed by IV maintenance dosing starting at Week 5 and then once every 2 weeks for 24 weeks. After 24 weeks of treatment, patients can continue crovalimab or switch from eculizumab to crovalimab if their physician determines this is in their best interest. The primary efficacy objective of COMMODORE 2 is to evaluate the noninferiority of crovalimab compared with eculizumab based on the co-primary endpoints of (1) the proportion of patients who achieve transfusion avoidance and (2) the proportion of patients with hemolysis control. Secondary efficacy objectives are to evaluate the noninferiority of crovalimab compared with eculizumab in regard to the (1) proportion of patients who experience breakthrough hemolysis, (2) proportion of patients who achieve stabilization of hemoglobin, and (3) mean change in fatigue, as assessed by the Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire. The safety objective is to evaluate the safety and tolerability of crovalimab compared with eculizumab based on the incidence and severity of adverse events, including infections (meningococcal meningitis and other infections), injection-site reactions, infusion-related reactions, hypersensitivity, and adverse events leading to study drug discontinuation. Pharmacokinetic, immunogenicity, biomarker, and health status utility objectives will also be assessed. Disclosures Kulasekararaj: Alexion Pharmaceuticals Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. He:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; LongBio Pharma: Consultancy, Research Funding. Munir:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; Alexion: Honoraria. Pu:SUNY Upstate Medical University: Current Employment; Pennsylvania State University: Patents & Royalties; F. Hoffmann-La Roche Ltd: Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Risitano:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biocryst: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Membership on an entity's Board of Directors or advisory committees; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; RA pharma: Research Funding. Röth:Roche: Consultancy, Honoraria, Research Funding; Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Biocryst: Consultancy, Honoraria. Sima:F. Hoffmann-La Roche Ltd/Genentech: Current Employment, Current equity holder in publicly-traded company, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Appius:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Sreckovic:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Vignal:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Nishimura:Alexion: Honoraria, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Abstract: The recurrent chromosome 16 inversion (inv(16)) in acute myeloid leukemia (AML) subtype M4Eo results in a fusion between CBFB and MYH11 genes, which encodes a chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain). We previously generated mouse CBFB-MYH11 knock-in models that mimic the human inv(16) AML and demonstrated that the CBFβ-SMMHC fusion protein blocks RUNX1 and CBFβ function during definitive hematopoiesis and plays a driving role in leukemogenesis. Our recent studies indicated that the C-terminus of CBFβ-SMMHC, which contains domains for multimerization and transcriptional repression, is important for leukemogenesis by CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain of CBFβ-SMMHC during hematopoiesis and leukemogenesis. Previous studies have dissected the assembly competence domain (ACD) of the CBFβ-SMMHC C-terminus to identify the critical amino acid residuals for multimerization (Zhang et al., Oncogene 25:7289, 2006). Among them, mutations in helices D and E are the ones that affect multimerization the most. Importantly, the helices D and E mutations do not interfere with the repression function of CBFβ-SMMHC. Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D & E in the ACD of the C-terminus (mDE) to determine the role of multimerization for the in vivo function of CBFβ-SMMHC. The embryonic hematopoietic phenotype in the mDE knockin embryos is very similar to what we have observed in the knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013), i.e., heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed hemorrhage in the central nervous system and died around E12.5, as seen in the full length CBFβ-SMMHC heterozygous knockin mice and the Cbfb-/- and Runx1-/- mice. Analysis of peripheral blood from adult Cbfb+/mDE mice showed decreased B cell population and increased T cell population, while the myeloid compartment was unchanged. Preliminary findings suggest that leukemogenesis is at least delayed in the Cbfb+/mDE mice as compared to mice expressing full-length CBFβ-SMMHC. Therefore the multimerization function of CBFβ-SMMHC is critical for its ability to induce defects in embryonic hematopoiesis and for leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Abstract: Among acute myeloid leukemia (AML) with cytogenetic abnormalities, core binding factor (CBF) leukemia acounts for 20-30% of adult AML, and 20-30% of pediatric AML. The chromosome 16 inversion (inv(16)), which results in a fusion gene CBFB -MYH11 and an encoded chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain), is observed primarily in AML subtype M4Eo. Using Cbfb-MYH11 knock-in mouse models we previously demonstrated that CBFβ-SMMHC needs its C terminal domains for leukemogenesis (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain at the C terminus of CBFβ-SMMHC for hematopoietic defects and leukemogenesis. Previous studies have shown that the C-terminal 29-residue assembly competent domain (ACD) is essential for multimerization of SMMHC. Within ACD, clustered point mutations in helices D and E specifically disrupts multimerization of CBFβ-SMMHC without interfering with the repression function of CBFβ-SMMHC (Zhang et al., Oncogene 25:7289, 2006). Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D and E (mDE) to study the role of the multimerization domain in vivo. Heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed complete blockage of definitive hematopoiesis, hemorrhage in the central nervous system and midgestation lethality, similar to the phenotype in Cbfb+/MYH11 mice and the Cbfb or Runx1 null mice. This phenotype is also similar to that in the homozygous knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). The fetal liver of E12.5 CbfbmDE/mDE embryos gave no colonies while the fetal liver of Cbfb+/mDE mice generated similar number of colonies as the WT controls. We further looked at the peripheral blood of E10.5 CbfbmDE/mDE embryos and found that the primitive hematopoiesis was not affected, while E10.5 Cbfb+/MYH11 embryos showed a developmental delay at this stage. Analysis of peripheral blood showed decreased B cell population in young adult Cbfb+/mDE mice, while the myeloid compartment was unchanged. In aged mice ( 〉 12 months), however, there was an increase of immature myeloid cells in the peripheral blood. Importantly, there was no leukemia development in the Cbfb+/mDE mice one year after ENU treatment (to induce cooperating mutations), while Cbfb+/MYH11 micedied of leukemia within 2 months of ENU treatment. Notably bone marrow cells in the Cbfb+/mDE and Cbfb+/MYH11 mice expressed their respective fusion proteins at similar levels. Overall our data suggest that the C terminal multimerization domain is required for the defects in primitive and definitive hematopoiesis caused by CBFβ-SMMHC, and the domain is essential for leukemogenesis by CBFβ-SMMHC. Further mechanistic studies of this domain may lead to new drug targets for treating inv(16) leukemia. For this purpose we have performed gene expression profiling with microarray and RNA-seq technologies, comparing gene expression changes in adult bone marrow c-Kit+ cells as well as embryonic primitive blood cells from Cbfb+/mDE and Cbfb+/MYH11 mice. Preliminary analysis indicates that the gene expression profile of the hematopoietic cells from the Cbfb+/mDE mice was much similar to that of Cbfb+/+ than Cbfb+/MYH11 mice. Validation and pathway analysis of those differentially expressed genes are ongoing and the results will be presented at the annual meeting. Disclosures No relevant conflicts of interest to declare.

Abstract: Purpose: To study the efficacy and side effects of humanized anti-CD19-CAR T-cell combined with multiple combination treatments in relapsed/refractory B-cell non-Hodgkin lymphoma. Methods: Thirty-eight relapsed/refractory B-cell non-Hodgkin lymphoma patients were enrolled in a clinical trial of anti-CD19 chimeric antigen receptor (CAR) modified T-cell expressing humanized anti-CD19 scFv and 4-1BB-CD3ζ costimulatory-activation domains therapy (ChiCTR1800018059) at the Department of Hematology in Tianjin First Center Hospital (Tianjin, China). Except for anti-CD19-CAR T-cell therapy, all the patients received multiple combination treatments before or at the same time of CAR-T cell therapy. The multiple combination treatments included anti-CD22-CAR T-cell, Programmed death 1 (PD-1) inhibitor, Bruton's tyrosine kinase (BTK) inhibitors, Lenalidomide, Advance high-dose chemotherapy and Radiotherapy. Patients were evaluated 2 months after the infusion. The side effects were observed in all the process of the combination treatments. Cytokine release syndrome (CRS) was graded according to the ASBMT Consensus Criteria. Results: The median time from infusion to cutoff day was 7 months (Range: 1 to 16 months). There were 37 patients included in the evaluation of the efficacy and side effects (Except for one patient with high tumor burden died of CRS 7 days after the infusion). The best overall response rate (ORR) was 75.6% (28/37). The complete response (CR) rate was 45.9% (17/37), the partail response (PR) rate was 29.7% (11/37), the stable disease (SD) rate was16.2% (6/37) and the disease progression (PD) rate was 8.1% (3/37). The grade of cytokine release syndrome (CRS) was Grade 1: 25/38, Grade 2: 9/38, Grade 3: 3/38, Grade 4: 0/38 and Grade 5: 1/38. Two patients died from disease progression within 60 days after infusion. One patient with high tumor burden who achieved PR at 14 days died from intestinal perforation after 25 days of infusion. The combination treatments of anti-CD22-CAR T-cell (4/38), treatment dose or 1/2 treatment dose of PD-1 inhibitor (5/38) or BTK inhibitors (3/38) therapy improved the curative effect of humanized anti-CD19-CAR T-cell therapy. Advance high-dose chemotherapy for patients (9/38) with high tumor burden before the anti-CD19-CAR T-cell therapy achived a satisfactory ORR (5/9) and Grade 1-2 CRS. No patients died from the advance and intensive chemotherapy. There were 6 patients with refractory lymphoma received radiotherapy before they enrolled in the anti-CD19-CAR T-cell therapy. There were 5 patients achived CR or PR except for one patient with high tumor burden died of CRS 7 days after the infusion (the IL-6 level was more than 1000 U/mL at day 3). But the side effects were significant in these 6 patients (Grade 1: 0/6, Grade 2: 2/6, Grade 3: 3/6, Grade 4: 0/6, Grade 5: 1/6). There was no efficacy improvement from the combination treatments of lenalidomide (14/38, 3 patients received advance high-dose chemotherapy meanwhile among the 14 patients), and the side effects were not significant. Conclusions: The multiple combination treatments combined with humanized anti-CD19-CAR T-cell therapy could improve the efficacy without increase side effects obviously. Disclosures No relevant conflicts of interest to declare.

Abstract: Protein disulfide isomerase (PDI) is a widely expressed oxidoreductase that catalyzes the rearrangement of intramolecular disulfide bonds during protein maturation in the endoplasmic reticulum. However, PDI can also be secreted from both platelets and endothelial cells during thrombus formation. Inhibition of extracellular PDI in the vasculature using neutralizing antibodies or bacitracin blocks thrombus formation in pre-clinical studies. Our previous experiments identified quercetin-3-rutinoside as an inhibitor of PDI and a potent antithrombotic. Recently, we performed a high-throughput screen of the MLSMR library through the Molecular Libraries Probe Production Centers Network (MLPCN) to identify more potent and selective inhibitors of PDI. We now compare the activity of the most potent lead, ML359, to previously described PDI inhibitors and assess its antithrombotic activity. ML359 inhibited the reductase activity of PDI in the insulin turbidometric assay with an IC50 of 0.3-0.6 microM. By comparison, quercetin-3-rutinoside and juniferdin inhibited PDI with IC50s of 6-10 microM, while bacitracin inhibited PDI reductase activity with an IC50 of 100 microM. ML359 demonstrated no confirmed activity at 〈 10 microM in any of the other of 380 biological assays in which it has been tested within the MLPCN. Additionally, ML359 was selective for PDI, failing to inhibit ERp5, ERp57, ERP72, thioredoxin or thioredoxin reductase activity. Inhibition of PDI by the ML359 was entirely reversible and it did not demonstrate toxicity in a HeLa cell assay. In preparation for pre-clinical studies, further evaluation of the biophysical properties of ML359 was performed. ML359 demonstrated acceptable solubility in PBS, human plasma and GSH. However, it showed poor stability in mouse plasma as well as in presence of liver microsomes. To optimize and improve these biophysical properties of the ML359, over 85 analogs of the lead were synthesized. Modulation of the ethyl substituent of the ester group with bulky isopropyl or t-butyl substituents increased the mouse plasma stability from ∼5% in ML359 to ∼75% and ∼95% in the analogs, respectively. These substitutions only minimally altered the potency of the compound (0.6-0.9 microM). The ML359 analogs demonstrated increased inhibitory activity in platelet aggregation assays, with inhibition at 30 microM increasing from 25% for ML359 to 97 and 100% for the isopropyl and t-butyl substituted analogs, respectively. The ability of ML359 to prevent thrombus formation in a laser injury mouse model was examined. Thrombus formation was monitored following laser injury of cremaster arterioles and the effect of ML359 infusion on platelet accumulation evaluated. ML359 exposure resulted in a significantly smaller thrombus that that observed in the animals infused with vehicle alone. ML359 and its active analogs have enhanced properties compared to the first generation PDI antagonists. These compounds are substantially more potent than previously described PDI inhibitors such as bacitracin and show significant potential as second generation inhibitors to probe PDI function in biological systems. ML359 analogs could ultimately be developed as antithrombotics that target PDI. Disclosures: No relevant conflicts of interest to declare.