Kurzfassung: Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.

Kurzfassung: Macrophages infiltrate and establish in developing organs from an early stage, often before these have become vascularised. Similarly, leukocytes in general can quickly migrate through tissues to any site of wounding. This unique capacity is rooted in their characteristic amoeboid motility, the genetic basis of which is poorly known. Trim33 (Tif1-γ), a nuclear protein that associates with specific DNA binding transcription factors to modulate gene expression, has been found mainly involved in hematopoiesis and gene regulation by TGF-ß. Here we have discovered that in Trim33 deficient zebrafish embryos, primitive macrophages are unable to colonize the central nervous system to become microglia. Moreover, both macrophages and neutrophils of Trim33 deficient embryos display a reduced basal mobility within interstitial tissues, and a profound lack of response to inflammatory recruitment signals, including local bacterial infections. Correlatively, Trim33 deficient mouse bone marrow derived macrophages display a strongly reduced three-dimensional amoeboid mobility in fibrous collagen gels. The transcriptional regulator Trim33 is thus revealed as essential for the navigation of macrophages and neutrophils towards developmental or inflammatory cues within vertebrate tissues.

Kurzfassung: The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c- myc under control of the zebrafish Rag2 promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c- myc – induced carcinogenesis.

Kurzfassung: Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was successful even when diluted 1000-fold, allowing easy identification of transgenic founders. Given its speed and low cost, we anticipate that the adoption of this method will streamline DNA isolation for zebrafish research.

Kurzfassung: Abstract 2129 Objectives: T cell lineage is an independent high risk factor in acute lymphoblastic leukemia (ALL). T-ALL requires high dose multi-agent chemotherapy, conferring many toxic side effects. T-ALL treatment therefore needs new, targeted agents that preserve or improve current treatment efficacy, yet cause fewer side effects than existing chemotherapeutic regimens. To identify such drugs, we pioneered an in vivo screen using transgenic zebrafish with T cell-specific green fluorescent protein (GFP) expression. We reasoned that immature T cells in 5-day-old zebrafish larvae are developmentally analogous to T-ALL lymphoblasts, and likely rely upon similar signaling pathways. Hence, compounds that specifically eliminate T cells in zebrafish larvae might likewise selectively target T-ALL cells. An added benefit of our in vivo screen is that drugs added to the water housing fish larvae must cross an epithelial barrier, can be metabolized by the liver, and can be renally cleared, characteristics not assessed in in vitro-based screening strategies. In addition, by using early larvae as our subjects, drugs lacking T cell specificity will likely impair normal development and/or survival, which we postulate is a predictor of unwanted toxicities. In these ways, our screen mimicked the scenario encountered in patients. Our use of the transgenic p56lck::EGFP zebrafish line facilitated rapid visual assessment of efficacy of a large number of compounds in 96-well format. Materials and Results: In a proof-of-principle experiment, we identified several known anti-T-ALL drugs, most prominently glucocorticoids, from the “Spectrum” library (MicroSource Discovery Systems, Inc., Gaylordsville, CT) of well-characterized compounds. We then screened 39,200 small molecules from the “ChemBridge DIVERSet” combinatorial chemistry library (ChemBridge Corp., San Diego, CA) for those active against zebrafish larval T cells. Of 20 novel “hits” identified, one compound, dubbed Lenaldekar (“LDK”), met additional prioritization criteria. LDK does not impair the cell cycle of developing zebrafish, is well tolerated and orally bioavailable with favorable pharmacokinetic properties in mice. In addition, pilot studies with LDK indicate it is efficacious in treating T-ALL in juvenile and adult fish from an established transgenic rag2::ER-human cMyc T-ALL model. LDK kills all of several murine T-lymphocytic malignancies, induces apoptosis in all human T-ALL lines tested, and shows some activity in human B-ALL lines. However, glioblastoma, colon carcinoma, melanoma, or immortalized human embryonic kidney cell lines are not affected by LDK, even at high concentration (up to 25μM). Using the recently established “phosflow” technique we measured phosphorylation status of key signaling proteins in permeabilized T- and B-ALL lines. Regardless of PTEN status, PDK1, AKT and mTOR downstream targets p4EBP1 and p70S6kinase were dephosphorylated by LDK treatment, as was the p65 subunit of NFκB on serine 529. Results were corroborated by conventional Western blots. However, phosphorylation of STAT3, STAT5, pERK1-2, and p38 were not affected by LDK. LDK's action is distinguished from other AKT/mTOR inhibitors by its lack of activity against AKT-dependent glioblastoma and melanoma cell lines, and its lack of effect on cell size. Finally, LDK decreased tumor burden of human T-ALL in murine xenografts. Conclusions: In view of its apparent lymphocyte selectivity, we posit that LDK modulates a pathway relatively unique to ALL (and immature lymphoblast) cells. This suggests that LDK can serve as a novel molecular tool for studies of normal and malignant lymphocyte biology. Moreover, with its favorable pharmacokinetics, apparent lack of toxicity, and in vivo efficacy in two vertebrate ALL models, LDK is an attractive molecule for development into a targeted treatment for ALL and perhaps other lymphocytic malignancies. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Background: To date, CAR T cell therapy has generally been limited to inpatient treatment at university medical centers. However, most patients (pts) in the US with R/R diffuse large B cell lymphoma receive therapy at non-university medical centers where outpatient delivery of cancer therapy is common. Infusion and management of CAR T cell therapies in the outpatient setting may lead to wider utilization in community/non-university centers and improve access. In TRANSCEND NHL 001, lisocabtagene maraleucel (liso-cel), an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at target doses of CD4+ and CD8+ CAR T cells, demonstrated efficacy as ≥3rd-line therapy in pts with R/R large B cell NHL. With fewer than half of pts developing cytokine release syndrome (CRS) and/or neurological events (NE) and its delayed onset (median 5 and 10 days, respectively; Abramson, ASCO 2018; 7505) outpatient treatment was allowed with hospitalization at the first sign of fever or neurological symptoms. We report on pts with R/R large B cell NHL who were treated with liso-cel in the outpatient setting in TRANSCEND NHL 001 (NCT02631044) and in two phase 2 studies assessing the safety and efficacy of liso-cel, as ≥3rd-line therapy (OUTREACH; NCT03744676) or as therapy for 2nd-line transplant noneligible (TNE) pts (PILOT; NCT03483103). Methods: Eligible pts had R/R large B cell NHL, adequate organ function, and prior systemic chemoimmunotherapy (TRANSCEND and OUTREACH: ≥2 prior lines of therapy and ECOG PS ≤1; PILOT: 1 prior line of therapy and deemed TNE for autologous hematopoietic stem cell transplantation based on ECOG PS, organ function, and/or age). After lymphodepletion with fludarabine/cyclophosphamide, liso-cel was administered at 1 of 3 dose levels (DL) (DL1 = 50 × 106, DL2 = 100 × 106, DL3 = 150 × 106 total CAR+ T cells) in TRANSCEND and at DL2 in OUTREACH and PILOT. All studies allowed outpatient treatment at non-university (OUTREACH) or at both university and non-university medical centers (TRANSCEND, PILOT). Outpatient treatment required pts to have a caregiver for 30 days post-liso-cel infusion, receive safety-monitoring education (recognizing critical adverse events; eg, fever), and to stay within 1 h travel to the site of care. Outpatient treatment was at the discretion of the investigator. All sites had a multidisciplinary CAR T cell therapy team and standard operating procedures for outpatient administration and toxicity monitoring. CRS (Lee criteria, 2014) and NEs (defined as related to liso-cel; CTCAE criteria) were managed in the hospital. Results: At data cutoff, 37 pts across studies had received liso-cel on study Day 1 and were monitored as outpatients, including pts ≥65 yr old and those with high tumor burden. Patient characteristics are shown in the Table. The most frequent grade ≥3 treatment-emergent AEs were cytopenias (neutropenia 43%, anemia 30%, thrombocytopenia 14%). Sixteen pts had any grade CRS and 12 had any grade NE (19 pts had CRS and/or NE). Severe CRS and/or NE occurred in only 2 pts and were reversible (Table). Three pts received tocilizumab and corticosteroids and 4 pts received corticosteroids alone for CRS and/or NE. No pts received tocilizumab alone. Twenty-two of the 37 pts (59%) required hospitalization at any time; all pts were from TRANSCEND or OUTREACH. Three of those pts (8%) were admitted on study Day 3 or earlier, all for CRS; 1 patient required ICU-level care (length of stay, 3 days). Median (range) time to hospitalization post treatment was 5 (2‒22) days and median (range) length of stay was 6 (2‒23) days. Fifteen (41%) of the 37 pts, including all 5 pts from PILOT, were not admitted to hospital in the first 29 days post liso-cel infusion. Across all 3 studies, most pts achieved an objective response, including CR (Table). Median time to peak CAR+ T cell expansion in each study was 10 days (range: 3-21 in TRANSCEND; 7-10 in OUTREACH; 7-10 in PILOT). Conclusions: A subset of pts with R/R large B cell NHL were successfully treated with liso-cel and monitored for CAR T cell-related toxicity in the outpatient setting, including elderly pts and pts with high tumor burden. Severe CRS and NEs occurred with low incidence. The number of early hospitalizations was low, and 41% of pts did not require hospitalization in the first month post liso-cel infusion. Most pts (65%) achieved an objective response. Updated data with longer follow-up will be presented. Disclosures Bachier: Sanofi: Speakers Bureau; Viracyte: Consultancy; Kadmon Corporation, LLC: Consultancy. Palomba:Noble Insights: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Hemedicus: Speakers Bureau; Merck & Co Inc.: Consultancy; Seres Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; STRAXIMM: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Evelo: Equity Ownership; MSK (IP for Juno and Seres): Patents & Royalties. Abramson:AbbVie Inc, Amgen Inc, Bayer HealthCare Pharmaceuticals, Celgene Corporation, EMD Serono Inc, Genentech, Gilead Sciences Inc, Janssen Biotech Inc, Juno Therapeutics, a Celgene Company, Karyopharm Therapeutics, Kite Pharma Inc, Merck, Novartis, Seattle Gen: Consultancy. Andreadis:Roche: Equity Ownership; Novartis: Research Funding; Celgene: Research Funding; Juno: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Kite: Consultancy; Genentech: Consultancy, Employment; Gilead: Consultancy; Jazz Pharmaceuticals: Consultancy. Sehgal:Juno/Celgene: Research Funding; Kite/Gilead: Research Funding; Merck: Research Funding. Hildebrandt:Juno Therapeutics: Equity Ownership; crispr therapeutics: Equity Ownership; CVS Health: Equity Ownership; Immunomedics: Equity Ownership; IDEXX laboratories: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Cellectis: Equity Ownership; Clovis Oncology: Equity Ownership; Aetna: Equity Ownership; Bluebird Bio: Equity Ownership; Celgene: Equity Ownership; Abbvie: Equity Ownership; Cardinal Health: Equity Ownership; Johnson & Johnson: Equity Ownership; Insys Therapeutics: Equity Ownership; Axim Biotechnologies: Equity Ownership; Axim Biotechnologies: Equity Ownership; Novartis: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Sangamo: Equity Ownership; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; Bayer: Equity Ownership; Scotts-Miracle: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Astellas: Other: Travel; Endocyte: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Novartis: Equity Ownership. Siddiqi:PCYC: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Kite, A Gilead Company: Research Funding; TG Therapeutics: Research Funding; Celgene: Research Funding; BeiGene: Research Funding; Juno Therapeutics: Consultancy, Other: travel support, Research Funding. Stevens:Astellas: Consultancy. Farazi:Juno Therapeutics/A Celgene Company: Employment. Kostic:Juno Therapeutics, a Celgene Company: Employment. Trede:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment. Lymp:Celgene Corporation: Employment, Equity Ownership. Thelen:Celgene Corporation: Employment. Ogasawara:Celgene Corporation: Employment, Equity Ownership. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; A2 Biotherapeutics: Honoraria, Other: Stock options ; BioLine RX, Gilead,Genentech,Novartis: Honoraria.