2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL

Experimental Hematology 2019;76 (Suppl): S42−S50

Short Talk Presentations 2000 - MONOLINEAGE ORIGIN OF RELAPSE FOLLOWING MULTILINEAGE DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIA Ross Dickins1, Steven Ngo1, Ethan Oxley1, Margherita Ghisi1, Mark McKenzie2, Maximilian Garwood1, Swathy Jayakrishnan1, Olivia Susanto1, Helen Mitchell1, Michael Hickey1, Andrew Perkins1, Benjamin Kile1, Ross Dickins1 1 Monash University, Melbourne, Australia; 2Walter and Eliza Hall Institute, Melbourne, Australia

transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.

Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite nearcomplete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.

2001 - LONG-TERM EX VIVO EXPANSION OF FUNCTIONAL HEMATOPOIETIC STEM CELLS Adam Wilkinson1, Reiko Ishida2, Misako Kikuchi2 , Kazuhiro Sudo3 , Maiko Morita2, Ralph Valentine Crisostomo1, Ryo Yamamoto1 , Kyle Loh1, Yukio Nakamura3, Motoo Watanabe2, Hiromitsu Nakauchi1, Satoshi Yamazaki2 1 Stanford University, Stanford, United States; 2 University of Tokyo, Tokyo, Japan; 3RIKEN BioResource Center, Tsukuba, Japan The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a »900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC

2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL Robert Signer, Miriama Kruta, Mary Jean Sunshine, Yunpeng Fu, Lorena Hidalgo San Jose UC San Diego, La Jolla, United States The inability to maintain and expand hematopoietic stem cells (HSCs) in culture represents a major barrier to their expanded use in cell-based therapies. We recently discovered that HSCs exhibit low protein synthesis in vivo, regardless of whether they are quiescent or undergoing self-renewing divisions. In the present study, we determined that cultured HSCs rapidly upregulated genes that promote translation and exhibited a »2000% increase in protein synthesis. Increased protein synthesis overwhelmed protein quality control systems within HSCs, caused an imbalance in protein homeostasis, and was associated with nuclear translocation of Hsf1. Hsf1 is the master regulator of the heat shock pathway, and induces transcription of heat shock proteins that coordinate proteotoxic stress response to maintain protein homeostasis. Inactive Hsf1 is typically sequestered in the cytoplasm, and is rarely seen in the nucleus of HSCs in vivo. Genetic deletion of Hsf1 exacerbated HSC depletion in vitro, but had little effect on HSC function in vivo. These data indicated that Hsf1 promotes ex vivo HSC maintenance, and raised the possibility that increasing Hsf1 activation could enhance HSC self-renewal. To test this, we developed a new serum- and stroma-free platform to culture purified HSCs. In 10-day cultures initiated with just 10 purified HSCs, small molecules that promoted Hsf1 nuclear translocation supported extensive proliferation and complete retention of long-term multilineage reconstituting activity in serial transplantation assays. The positive effect of these small molecules on HSC growth was completely ablated in

Short Talk Presentations / Experimental Hematology 2019;76 (Suppl): S42−S50 the absence of Hsf1. At the molecular level, Hsf1 activation reduced the unfolded protein load and partially rebalanced protein homeostasis in HSCs. These findings indicate that maintaining protein homeostasis is a key factor in promoting ex vivo HSC self-renewal.

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2004 - NOVEL PRO-INFLAMMATORY AGM-ASSOCIATED MACROPHAGES ARE INVOLVED IN EMBRYONIC DEVELOPMENT OF HEMATOPOIETIC STEM CELLS Samanta Mariani1, Zhuan Li2, Siobhan Rice3, Stamatina Fragkogianni4, Carsten Krieg5, Chris Vink1, Jeffrey Pollard1, Elaine Dzierzak1 1 The University of Edinburgh, Edinburgh, United Kingdom; 2 Guangzhou University, Guangzhou, China (People’s Republic); 3 The Queen’s College, Oxford, Oxford, United Kingdom; 4 Memorial Sloan Kettering Cancer Center, New York, United States; 5Medical University of South Carolina, Charleston, United States Hematopoietic stem cells (HSCs) are responsible for the life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors have been implicated in regulating HSC development, but it is unclear what cells in the mouse embryonic aorta-gonad-mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles and they are involved in bone marrow hematopoiesis. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our mass cytometry (CyTOF) results indicate the absence of lymphoid cells and mature neutrophils in E10.5 mouse AGM, while two abundant myeloid cell types are present − mannose-receptor positive AGM-associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1 and it occurs just before the time of HSC generation. Differently from mannose-receptor positive adult macrophages, AGM-aM express a pro-inflammatory signature. They localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult-repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM-associated macrophages in regulating the embryonic development of HSCs.

2003 - ACUTE MYELOID LEUKEMIA CELLS SEIZE SEROTONIN SIGNALING IN OSTEOBLASTS TO ENGRAFT AND PROLIFERATE Marta Galan-Diez1,2, Junfei Zhao3, Raul Rabadan3, Stavroula Kousteni4 1 Columbia University, Department of Physiology and Cellular Biophysics, New York, United States; 2 College of Physicians and Surgeons, New York, United States; 3Columbia University, New York, United States; 4Columbia University, New York, United States Patients with myelodysplasia or acute myeloid leukemia (AML) show decreased osteoblast numbers, and this is reproduced in mouse models of acute leukemia. Manipulating the osteoblast pool directly affects leukemia progression: osteoblast ablation increases leukemia burden, while maintenance of osteoblast numbers decreases it and prolongs survival. Here we show that the protective effect of osteoblasts against leukemia does not rely on a certain threshold of osteoblast numbers but rather requires a specific signaling pathway. Treatment of leukemic mice with parathyroid hormone, which increases osteoblast numbers, had no effect in leukemia progression. In contrast, maintaining the osteoblast pool in leukemic mice by decreasing the synthesis of gut-derived serotonin, reduced AML burden and prolonged survival. Using the human-AML MLL-AF9 model, we examined whether deletion of one of the main serotonin receptors expressed in osteoblasts affects leukemia progression. Global deletion of Htr1b in mice prevented leukemia. While removal of Htr1b in LepR+ positive mesenchymal stem cells had no effect in leukemia progression, its deletion in Col1a1-expressing osteoblast precursors significantly prevented lethality in the mutant mice. In order to dissect the crosstalk between leukemia-cells and osteoblasts, we co-cultured primary-human osteoblasts and AML cells. RNAseq analysis identified a signature of chemoattractants and pro-inflammatory cytokines secreted by osteoblasts upon exposure to AML cells, suggesting that the protective mechanism exerted by osteoblasts activates the anti-tumor immune response against leukemia. In summary, our data suggest that AML cells activate serotonin signaling in osteoblasts to promote their engraftment and expansion. Targeting this pathway may render the niche hostile to leukemia by enhancing the anti-tumor immune response elicited by osteoblasts.

2005 - GATA3 PROMOTES THE ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION VIA REGULATION OF THE CELL CYCLE Katrin Ottersbach1, Nada Zaidan2 , Evangelia Diamanti3, Emma Shaw1, Nicola Wilson3, Berthold Gottgens3 1 University of Edinburgh, Edinburgh, United Kingdom; 2King Abdullah International Medical Research Centre, Riyadh, Saudi Arabia; 3University of Cambridge, Cambridge, United Kingdom Hematopoietic stem cells (HSCs) are known to emerge in the dorsal aorta at E10.5 of mouse embryo development. They derive from hemogenic endothelial cells via a process known as endothelial-to-hematopoietic transition (EHT), which comprises a number of intermediate steps and the molecular details of which have not been fully characterized. Our lab has previously demonstrated that embryos deficient for the transcription factor Gata3 have a defect in HSC generation in the dorsal aorta. This was linked to a lack of catecholamine synthesis in these embryos, which demonstrated that neurotransmitters released from the co-developing sympathetic nervous system promote embryonic HSC production. We now have evidence that Gata3 plays an additional role in EHT. Gata3 expression was found to enrich for hemogenic endothelial cell activity, and an endothelial-specific knockout of Gata3 severely impairs HSC generation in the dorsal aorta. Furthermore, with the use of co-aggregation assays developed in the Medvinsky lab, we were able to determine that Gata3 is expressed in the pro-HSC and pre-HSC type I stage, but is downregulated thereafter. RNA-Seq analysis of Gata3-positive and negative cell populations highlighted a cell cycle signature, with cell cycle inhibitors, such as p57Kip2, specifically enriched in Gata3-expressing cells. This suggests that a cell cycle arrest may be required for endothelial cells to complete the transition to a hematopoietic fate, which is supported by our preliminary results with embryos deficient for p57Kip2. Our results thus provide further insights into the molecular mechanisms of HSC generation from endothelial cells.