ALBERT

Beschreibung: The asymmetric units of the title compounds both contain one nonplanar molecule. In 4-benzyl-6-phenyl-4,5-dihydropyridazin-3(2H)-one, C17H14N2O, (I), the phenyl and pyridazine rings are twisted with respect to each other, making a dihedral angle of 46.69 (9)°; the phenyl ring of the benzyl group is nearly perpendicular to the plane of the pyridazine ring, the dihedral angle being 78.31 (10)°. In methyl 2-[5-(2,6-dichlorobenzyl)-6-oxo-3-phenyl-1,4,5,6-tetrahydropyridazin-1-yl]acetate, C20H16Cl2N2O3, (II), the phenyl and pyridazine rings are twisted with respect to each other, making a dihedral angle of 21.76 (18)°, whereas the phenyl ring of the dichlorobenzyl group is inclined to the pyridazine ring by 79.61 (19)°. In the crystal structure of (I), pairs of N—H...O hydrogen bonds link the molecules into inversion dimers with an R 2 2(8) ring motif. In the crystal structure of (II), C—H...O hydrogen bonds generate dimers with R 1 2(7), R 2 2(16) and R 2 2(18) ring motifs. The Hirshfeld surface analyses of compound (I) suggests that the most significant contributions to the crystal packing are by H...H (48.2%), C...H/H...C (29.9%) and O...H/H...O (8.9%) contacts. For compound (II), H...H (34.4%), C...H/H...C (21.3%) and O...H/H...O (16.5%) interactions are the most important contributions.

Beschreibung: An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Beschreibung: Background: Quizartinib is a once-daily, oral, highly potent and selective FLT3 inhibitor with proven single-agent activity and a manageable safety profile in FLT3-ITD R/R AML. In the global, phase 3 QuANTUM-R study, quizartinib demonstrated a significant survival benefit over salvage chemotherapy(Cortes, et al. Lancet Oncol, 2019; NCT02039726). Additional support for the safety profile of quizartinib at the proposed dosing regimen (60 mg QD with a 30-mg starting dose) is provided by a pooled analysis of over 600 patients with R/R AML. Methods: We pooled data from four studies: one phase 3 (QuANTUM-R), two phase 2, (ACC220-002 and 2689-CL-2004; Cortes, et al. Lancet Oncol, 2018; Cortes, et al. Blood, 2018), and one phase 1 (CP0001: only patients assigned to continuous daily dosing regimens are included in this analysis; Cortes, et al. J Clin Oncol, 2013) to provide an integrated safety summary of quizartinib monotherapy for the treatment of R/R AML. Treatment-emergent adverse events (TEAEs), on-treatment deaths, and adverse events of special interest (AESIs; QT prolongation and potential ventricular arrhythmias, hepatic disorders, and sequelae of cytopenias such as infections and bleeding) with quizartinib were analyzed. Results: The pooled data set included 673 patients; 51% were male, and median age was 59 years. Thirty-eight patients were assigned to quizartinib 30 mg, 277 to 60 mg, and 358 to 〉60 mg up to 300 mg. Overall, median treatment duration was 79 days (range, 1-1296 days) and was longest in the 60-mg dose group (95 days) vs the 〉60-mg (70.5 days) and 30-mg (66 days) groups. The overall TEAE profile was consistent with the results from the phase 3 QuANTUM-R study. Most on-treatment deaths (215 of 599 evaluable patients [36%]; defined as taking place between the first dose and ≤30 days after the last dose) were attributed to AML disease progression (130/599 [22%]), followed by TEAEs (83/599 [14%]), which were predominantly respiratory tract infections and events related to sepsis. TEAEs leading to discontinuation of quizartinib occurred in 173/673 patients (26%). In the 60-mg dose group (quizartinib proposed dosing regimen), the only TEAE associated with discontinuation in 〉2% of patients was pneumonia (6/277 patients [2%]). The most frequently reported grade ≥3 TEAEs were febrile neutropenia (240/673 [36%]), anemia (188/673 [28%]) and thrombocytopenia (182/673 [27%]) in the hematologic category and pneumonia (119/673 [18%]) and sepsis/septic shock (124/673 [18%]) in the nonhematologic category (Table 1A). In the 60-mg dose group, the most frequent AESI was infection (210/277 [76%]; Table 1B). Epistaxis and petechiae, mainly grade 1/2, were the most frequently reported hemorrhage TEAEs. TEAEs in the hemorrhage category occurred more frequently in the 〉60-mg dose group than the 60-mg dose group (215/358 [60%] and 136/277 [49%], respectively). The most frequent serious hemorrhage TEAEs were gastrointestinal hemorrhages (15/673 [2%]) and intracranial hemorrhage (10/673 [2%]). QTcF prolongation 〉500 ms (grade 3) occurred in 75/673 patients (11%), mostly in those receiving quizartinib 〉60 mg (n = 64) (Table 1C). In the 60-mg dose group, QTcF prolongation 〉500 ms occurred in 9/277 patients (3%). The median time to onset of QTcF prolongation 〉500 ms was shorter in the 〉60-mg dose group than in the 60-mg dose group (9 and 46 days, respectively). Arrhythmias potentially related to QTcF prolongation were infrequent; one event of torsade de pointes, and one event of fatal cardiac arrest (both at doses 〉60 mg) were observed. No dose effects were seen with hepatic disorders, cardiac arrhythmias, or cardiac failure AESI categories. Conclusions: The overall safety profile of quizartinib in this R/R AML pooled analysis is consistent with that observed in QuANTUM-R. A reduction in the incidence of QTcF prolongation was noted with lower doses of quizartinib (30 or 60 mg vs 〉60 mg). There was also a reduced incidence of gastrointestinal symptoms, infections, and bleeding at lower doses of quizartinib (30 or 60 mg vs 〉60 mg). Results from this pooled analysis demonstrate that quizartinib is well tolerated at the proposed dosing regimen (60 mg QD with a 30-mg starting dose) in patients with R/R AML. Disclosures Cortes: BiolineRx: Consultancy; Biopath Holdings: Consultancy, Honoraria; Takeda: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Sun Pharma: Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Ganguly:Kite Pharma: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Seattle Genetics: Speakers Bureau; Daiichi Sankyo: Research Funding. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding. Levis:FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding. Martinelli:Daiichi Sankyo: Consultancy, Honoraria; Amgen: Consultancy, Other: trial grant; Pfizer: Consultancy, Other: trial grant; Ariad: Consultancy, Other: trial grant; Incyte: Consultancy, Other: trial grant; Roche: Consultancy, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Janssen: Consultancy, Other: trial grant; Novartis: Consultancy, Other: trial grant; Abbvie: Consultancy, Honoraria, Other: trial grant. Perl:Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.. Russell:Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; Pfizer Inc: Consultancy, Honoraria, Speakers Bureau. Choi:Daiichi Sankyo: Employment. Mendell:Daiichi Sankyo, Inc.: Employment. Namuyinga:Daiichi Sankyo: Employment. Pham:Daiichi Sankyo: Employment. Said:Daiichi Sankyo: Employment. Wang:Daiichi Sankyo: Employment. Mitov:Daiichi Sankyo UK Ltd: Employment. Kim:Daiichi Sankyo: Employment. Khaled:Alexion: Consultancy, Speakers Bureau; Daiichi Sankyo: Other: Travel support; Omeros: Consultancy.

Beschreibung: Background: Daily CBC (Complete blood count) and CMP (Complete metabolic profile) have been drawn on our patients with high-grade lymphoma admitted for inpatient chemotherapy with R-EPOCH (Rituximab, Prednisone, Vincristine, Cyclophosphamide, Doxorubicin) which is given over 5 days and R-ICE (Rituximab, Ifosfamide, Carboplatin, Etoposide) which is given over 3 day period. We conducted a retrospective study at our center looking to find if daily laboratory, other than day 1 laboratory, will have an impact on decisions regarding 1) change in chemotherapy dose or schedule 2) requirement of Blood products transfusion 3) cost effectiveness. Methods: We did retrospective review of 101 admissions between 2010-2017. We documented how frequent CBC and CMP were obtained during patient admissions. We documented any change in chemotherapy schedule or dose, or any need for blood product transfusion based on results of laboratory obtained starting day 2 of cycle 2. We excluded day 1 given that its results are required to proceed with chemotherapy. Also we excluded induction cycle 1. We documented cost of laboratory obtained other than day 1. Results: Daily CBC and CMP did not lead to any significant changes in plan of care of chemotherapy regimen and only lead to blood transfusions in 4.1% of patients on R-EPOCH and 6% of patients on R-ICE regimens. The cost associated with daily CBC and CMP was $1725 for R-EPOCH regimen and $1339 for R-ICE regimen. Conclusion: We recommend only day 1 CBC/CMP for patients admitted for R-EPOCH and R-ICE chemotherapy starting cycle 2. Daily CBC/CMP didn't lead to any significant changes on the plan of care and added more cost. Table Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction: Post-translational histone modifications directly modify chromatin structure to influence a wide variety of cellular events including gene expression, DNA replication and repair, and cell cycle control. While histone lysine methylation can confer either transcriptionally active or repressive states, the symmetric dimethylation of arginine residues on histone tails is generally associated with transcriptional repression. Overexpression and dysregulation of PRMT5, the major type II protein arginine methyltransferase, has been shown to drive cellular proliferation and survival of multiple cancer types including mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma associated with poor prognosis. Our work has shown that PRMT5 drives the symmetric dimethylation of arginine (histones H3R8 and H4R3) and attenuates RB/E2F regulatory pathways leading to the expression and activation of the PRC2 lysine methylation programs. We have previously shown that PRMT5 promotes constitutive expression of the WNT/β-CATENIN target genes (MYC, CCND1, and SURVIVIN) via the epigenetic repression of AXIN2 and WIF1 regulatory genes. This PRMT5 mediated epigenetic program allows for constitutive activation of WNT and PI3/AKT downstream pathways relevant to MCL growth and survival. The inhibition of PRMT5 triggers histone deacetylation and H3K4me3 demethylation on promoters of β-CATENIN target genes. H3K4 is modified by several enzymes containing SET domains including the SETD7 and MLL1 proteins. We hypothesized that PRMT5 inhibition, through its ability to repress AKT phosphorylation, would regulate SETD7 and MLL1 activity and regulate downstream pathways relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived MCL cell lines and primary lymphoma tumor cells was achieved with sh-PRMT5 lentivirus (or sh-GFP control) and utilization of a selective small molecule, SAM-competitive PRMT5 inhibitor (PRT382). Gene and protein expression was monitored by reverse transcription (RT) real time PCR and western immunoblotting, respectively. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. Interactions within the transcriptional complex were examined by co-immunoprecipitation. Cellular growth and apoptosis was assessed by proliferation assays and FACS analysis. Results: Inhibition of PRMT5 by shRNA-mediated knock down or treatment with PRT382 (Prelude Therapeutics) reduced H3K4me2 in MCL cells via indirect epigenetic repression of SETD7. ChIP-PCR studies showed PRMT5 to be recruited to the SETD7 promoter, suggesting that the activity of PRMT5 promoted the transcription of this lysine methyltransferase. Our findings show that reduced phosphor-AKT by PRMT5 inhibition inhibits dimerization of the MLL1 complex leading to dissociation of MLL1 from the transcriptional activation complex and decreased H3K4me1 and H3K4me3. PRMT5 inhibition led to dissociation of the BCL9- Pygopus-MLL1 transcriptional activating complex and to assembly of repressive LSD1 (the histone demethylase affecting H3K4me3)-HDAC2 (the histone lysine deacetylase) containing complexes. Furthermore, PRMT5 inhibition led to differentially enhanced recruitment of this repressive LSD1/HDAC2 complex and decreased H3K9Ac and H3K14Ac epigenetic marks on promoters of β-CATENIN target genes. PRMT5 inhibition regulates lysine methylation at H3K4 through epigenetic silencing of SETD7 and MLL1 activity, as well as histone H3 acetylation driven by histone acetyltransferase KAT2A. ChIP-Seq studies examining SETD7 and PRC2 recruitment in context of PRMT5 inhibition are currently underway. Conclusions: Our observations show that dysregulated PRMT5 activity acts as a master epigenetic regulator affecting arginine and lysine histone marks. PRMT5 can act directly to repress target tumor suppressor genes while simultaneously indirectly activating SETD7 and MLL1 to drive transcriptional activation of key oncogenes that promote the proliferation and survival of MCL. These results support the notion that inhibiting PRMT5 leads to erasure of repressive histone arginine and lysine marks and promote the restoration of normal growth and survival checkpoints in this disease. Disclosures Scherle: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Beschreibung: Introduction: Chronic immune thrombocytopenia (cITP) is a common, acquired, autoimmune disorder resulting in abnormally low blood platelet counts (