Kurzfassung: Platelets are major delivery vehicles for pro- and antiangiogenic growth factors. During the formation of new blood vessels, platelet-deployed factors support the recruitment and differentiation of bone marrow–derived cells. Therapeutic manipulation of the site-specific deployment of these factors by platelets may be used to inhibit tumor growth or promote tissue restoration.

Kurzfassung: In myeloid malignancies, presence of 'multi-hit' TP53 mutations is associated with lack of response to conventional therapy and dismal outcomes, particularly when found in combination with a complex karyotype. Therefore, it is crucial to understand the biological basis of TP53-mutant driven clonal evolution, suppression of antecedent clones and eventual disease transformation to inform the development of more effective therapies. Myeloproliferative neoplasms (MPN) represent an ideal tractable disease model to study this process, as progression to secondary acute myeloid leukemia (sAML) frequently occurs through the acquisition of TP53 missense mutations. To characterize tumor phylogenies, cellular hierarchies and molecular features of TP53-driven transformation, we performed single-cell multi-omic TARGET-seq analysis (PMID: 33377019 & 30765193) of 22116 hematopoietic stem and progenitor cells (HSPCs) from 35 donors and 40 timepoints (controls, MPN in chronic phase, pre-AML and TP53-mutated sAML; Figure1a). TARGET-seq uniquely enables single-cell mutation analysis with allelic resolution with parallel transcriptomic and cell-surface proteomic readouts. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type alleles upon transformation, including parallel evolution of separate TP53 "multi-hit" subclones in the same patient (n=4/14) and JAK2-negative progression (n=2/14). Complex clonal evolution driven by chromosomal abnormalities (CAs) was present in all patients and TP53 multi-hit HSPCs without CAs were rarely observed. Subclones with recurrent CA such as monosomy 7 showed upregulation of RAS-associated transcription and preferentially expanded in xenograft models. Together, these data indicate that TP53 missense mutation, loss of TP53 wild-type allele and cytogenetic evolution are collectively required for leukemic stem cell (LSC) expansion. Integrated transcriptomic analysis of sAML samples (Figure1b) revealed three major populations: (1) a TP53-mutant cluster (Figure1c) characterized by an erythroid signature (e.g. KLF1, GATA1, GYPA; an unexpected finding as no cases showed diagnostic features of erythroid leukemia), (2) an LSC TP53-mutant cluster (Figure1d) and (3) a TP53-WT preleukemic cluster (Figure1e). The LSC cluster showed dysregulation of key stem cell regulators, from which we derived a novel 48-gene LSC score with prognostic impact in an independent AML cohort (HR=3.13; Figure1f). Importantly, this score was predictive of outcome irrespective of TP53 status for both de novo and sAML, demonstrating its broader potential clinical utility. TARGET-seq analysis uniquely allowed us to characterize rare TP53-WT preleukemic cells (preLSCs), which were almost exclusively confined to the immunophenotypic lineage-CD34+CD38-CD90+CD45RA- HSC compartment. PreLSC from sAML samples presented increased stemness, increased quiescence, aberrant inflammatory signaling and differentiation defects (Figure1g) as compared to HSCs from normal or MPN donors, both at the transcriptional and functional levels through in vitro long-term and short-term cultures. This indicates cell-extrinsic suppression of residual TP53-WT hematopoiesis. Longitudinal analysis of TP53-heterozygous mutant HSPCs at different stages of disease evolution (Figure1a) revealed that aberrant inflammatory signalling (e.g. BST2, IFITM1, IFITM3) in the genetic ancestors of TP53 "multi-hit" LSCs, but not the presence of TP53-mutations alone, was predictive of subsequent transformation. In a mouse model system, TP53-mutant cells challenged with sustained inflammatory stimuli acquired a mean 3-fold competitive advantage in WT: TP53 R172H/+chimeras. This indicates that pro-inflammatory cues from the tumour microenvironment promote fitness advantage of TP53-mutant cells whilst supressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis of the genetic, cellular and molecular landscape of TP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia (Figure1h). Since TP53 is the most commonly mutated gene in human cancer, we anticipate these findings will be of broader relevance to many other cancer types. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment. Drummond: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mead: Abbvie: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau.

Kurzfassung: The hierarchical model of hematopoiesis posits that hematopoietic stem and progenitor cells produce common myeloid progenitors (CMP). CMP can become granulocyte/monocyte progenitors (GMP) or bipotential megakaryocyte/erythroid progenitors (MEP). MEP can produce megakaryocytic (Mk) or erythroid (Ery) cells. However, we and others have shown that early mouse and human progenitor populations express many Mk genes (Heuston, Epig. Chrom., 2016), while single cell studies have identified lineage-specific colony forming cells in progenitor populations thought to be multipotent (Psaila, Genome Biol., 2016). To identify the earliest mouse Ery and Mk cells, we performed single cell RNASeq on 10000 stem and progenitor cells (Lin-Sca1+Kit+), 12000 CMP (Lin-Sca1-Kit+CD16/32-CD34+), 6000 MEP (Lin-Sca1-Kit+CD16/32-CD34-) and 8000 GMP (Lin-Sca1-Kit+CD16/32+CD34+). TSNE analysis of expression in the 4 populations identified 33 clusters, which were correlated to biological functions using gene set enrichment analysis. In LSK, no cells with an Ery RNA profile were found, while 56% of cells co-expressed Mk-associated (e.g., Meis1, Fli1) and lymphoid genes. In CMP, 12% of the cells co-expressed Ery (e.g., Gata1, Fog1) and Mk (e.g., Pf4, Cd41) genes, while 23% had an Mk-specific profile (e.g., Fli1, Cd41) enriched for platelet biology processes (p & lt; 3E-18). Unlike traditional models, over 94% of MEP had Ery RNA profiles enriched for ribosome synthesis and heme-biology processes (p & lt; 4E-10). To establish developmental relationships, we performed pseudotime analysis using the Monocle and Scanpy software packages. These programs model differentiation by mapping similar transcriptomes together. Map nodes indicate lineage commitment points and cells further from a node are more differentiated. Combined analysis of LSK, CMP, and MEP generated a model with a single node and 2 trajectories. LSK with Mk and lymphoid RNA profiles diverged at the node, as did 14% of CMP. 31% of CMP with an Mk RNA profile were downstream of the node. Further downstream were cells with mixed Ery/Mk profiles, and furthest from the node were MEP with Ery profiles. A separate pseudotime analysis of CMP only 2 trajectories: one with decreasing Mk- and increasing Ery RNA profiles, and a second with an early Mk endomitotic RNA profile. Pseudotime analysis of MEP only identified a linear trajectory: cells at one end expressed early Ery RNA profiles, and cells at the other end had RNA profiles similar to those of burst-forming unit-erythroid (BFU-E). We generated a predictive set of RNAs for each TSNE cluster. We used index-sorting with 11 markers (Kit, Sca1, CD34, CD16/32, CD36, CD41, CD48, CD123, CD150, CD9, Flk2) to isolate single cells for custom high-throughput multiplex qPCR. This allowed confirmation of cell frequency within TSNE clusters while identifying surface markers for prospective isolation of cell subsets. We focused on 2 populations: CMP-E, which had an Ery RNA profile (10% of clustered CMP and 12% of CMP in the qPCR assay), and CMP-MkE, which had Mk and Ery RNA profiles (12% of clustered CMP and 13% of CMP in the qPCR assay). We prospectively isolated CMP-E and CMP-MkE to compare RNASeq profiles, ATACSeq profiles, and colony forming ability against those of bulk CMP, Ery, and Mk. In CMP-E, 54% of RNAs were expressed in both CMP and ERY, while 41% were expressed only in CMP (p & lt; 6E-72). In contrast, 41% of CMP-E ATACSeq peaks were present in CMP and ERY, while 57% of CMP-E peaks were present only in CMP (p & lt; 1E-3). We conclude that in CMP-E, the RNASeq profile is more erythroid than the ATACSeq profile. In CMP-MkE, 89% of RNAs were expressed in both CMP and Mk, while 7% were expressed only in CMP (p & lt; 8E-190). Likewise, 88% of CMP-MkE ATACSeq peaks were present in both CMP and Mk, while 3% were present only in CMP (p & lt; 1E-3). We conclude that in CMP-MkE, the RNASeq and ATACSeq profiles are equivalent. In soft agar assays, 21% of CMP-E and 3% of CMP-MkE colonies contained BFU-E, compared to 9% of control colonies. We conclude that the CMP-E and CMP-MkE populations are skewed towards the ERY and MK lineages, but are not erythro-megakaryocyte restricted. Our data support a model in which there are two megakaryocyte precursor populations and no erythroid populations in LSK. A third megakaryocyte population in CMP gives rise to erythroid cells. Finally, our data show that transcriptional changes precede chromatin accessibility changes in the earliest erythroid cells. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Identification and prospective isolation of EEP and LEP from human bone marrow (BM) facilitates the study of erythropoiesis. Quantitative and qualitative defects in EP underpinning erythropoietic failure in DBA are restored in steroid-responsive (SR) patients.

Kurzfassung: Abstract 470 Introduction: Specialized microenvironments, or niches, of the bone marrow (BM) maintain and regulate physiological haematopoietic stem/progenitor cells and have been implicated as areas of preferential engraftment and ‘sanctuary sites' for leukaemic cells and metastasizing cells of solid tumours. The cellular and molecular factors that comprise the BM vascular niche have not been well described. Megakaryocytes (MKs) reside in close association with the BM sinusoidal endothelium. The aim of this study was to investigate whether MKs play a role in the homing and engraftment of malignant cells in the BM. Methods: C57Bl/6 wild type and thrombopoietin (TPO)-/- mice (which have 〈 10% of the normal number of MKs and platelets) received flank or tail vein injections of the syngeneic B16-F10 melanoma or EL4 lymphoma cell lines fluorescently labelled with mCherry or GFP. The MK-vascular niche was examined using citrulline, MECA32, and TSP1 immunostaining. MKs were cultured from the Lin-Sca1+cKit+-enriched fraction of BM cells from flushed femurs and tibias, using 50ng/ml TPO + 20ng/ml SCF. Costar transwell co-culture plates and Neuroprobe chemotaxis chambers were used; cellular proliferation was assessed using the CellTitre MTS assay. RNA was extracted from flushed BM cells and in vitro cell cultures using Qiagen RNeasy columns/Trizol and gene expression was analyzed by RT-qPCR. Results: In wild type mice, the majority of BM sinusoids were surrounded by one or more large MKs forming the MK-vascular niche, with MKs tightly abutting the vascular endothelium. In TPO-/- mice, MKs were largely absent from the BM, blood vessels appeared more tortuous and the mean vessel diameter in the BM was significantly larger than in wild type mice (P 〈 0.01). Expression of the angiogenic regulatory proteins platelet factor 4 (PF4) and thrombospondin 1 (TSP1) was markedly lower in BM from TPO-/- mice than wild type; expression of VEGF and TGFb was also reduced. Together these findings suggest that MKs support the integrity of the vascular niche and that homeostasis of the niche may be disrupted in the absence of MKs. Wild type mice injected with either B16-F10 melanoma or EL4 lymphoma had increased numbers of MKs and a larger mean vessel diameter. Although there was no increase in platelet count, the mean platelet volume was significantly increased by day 18 (p=0.002), suggesting increased thrombopoiesis. Furthermore, there was a linear decrease in PF4 in response to tumour, reaching a 3-fold reduction by late-stage tumour growth. This finding was consistent with the increase in MK-vascular niches as PF4 normally acts as an autocrine inhibitor of megakaryopoiesis and inhibits endothelial cell proliferation and migration. Consistent with a modulatory effect of MKs and/or the MK-vascular niche on tumour phenotype, tumour growth in TPO-/- mice was markedly retarded and there was reduced metastasis to the BM and lung. To investigate the mechanism of these effects, MK-conditioned medium (MCM) was added to in vitro cultures of B16 cells. MCM significantly enhanced the proliferation rate of B16 melanoma cells (P 〈 0.001). Further, MCM was highly chemotactic for B16 cells (P 〈 0.001). This effect was found to be mediated by pertussis toxin-sensitive Gi-protein receptors and reduced but not entirely abrogated in the absence of TSP1 (using MCM generated from TSP1-/- mice). To investigate the interactions between tumour cells and MKs, MKs were cocultured with B16 cells. Coculture increased MK expression of proangiogenic factors VEGF and TGFb while cocultured B16 cells displayed increased expression of alpha integrins, a4, a5 and a6. Moreover, coculturing B16 cells with MKs prior to tail vein injection enhanced tumour cell engraftment in the lung. A pilot study of BM trephine biopsies from 8 patients with carcinoma (breast, lung, prostate, bladder and kidney) supported these preclinical findings. MKs in 3/3 patients with BM metastasis and 3/5 patients without BM metastasis showed a variable excess of MKs, some in loose clusters, with abnormal morphology and localization. Conclusions: These findings suggest that MKs contribute to the integrity and functionality of the BM vascular niche in homeostasis and in malignancy, and that cellular/molecular cross-talk between MKs and tumour cells at the vascular niche may promote metastasis. Targeting these interactions may be a useful as adjunctive therapy to prevent dissemination of cancer to the BM. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Intracranial hemorrhage (ICH) is a rare but devastating complication of childhood immune thrombocytopenia purpura (ITP). A survey of ICH from 1987 to 2000 identified cases of ICH in childhood ITP in the United States. Forty patients with ICH and 80 matched ITP control subjects were accrued. The estimated incidence of ICH was 0.19% to 0.78%. Platelet counts were less than 20 × 109/L in 90% and less than 10 × 109/L in 75% of children with ICH. Eighteen (45%) children developed ICH within 7 days of diagnosis of ITP; for 10 of these, ICH was the presenting feature of ITP. Twelve (30%) children had chronic ITP. Head trauma and hematuria were the most prominent features associated with ICH, identified in 33% and 22.5% of the patients with ICH and 1 and none of the controls (both P 〈 .001). Bleeding beyond petechiae and ecchymoses was also linked to ICH. Mortality was 25%; a further 25% had neurologic sequelae. Strategies by which high-risk children could be identified were considered, and the costs of preventive combination treatment were estimated. Children with severe thrombocytopenia plus head trauma and/or hematuria appeared to be at particularly high risk of ICH. Aggressive treatment of these children may be appropriate.

Kurzfassung: INTRODUCTION Eltrombopag (EP) is an orally bioavailable thrombopoietin receptor agonist developed to increase platelet production in a range of conditions associated with thrombocytopaenia. The finding that EP, in addition to increasing platelet counts in myelodysplastic syndromes (MDS), also slowed progression to acute myeloid leukemia has been linked to antiproliferative effects potentially mediated by chelation of labile intracellular iron pools in leukaemia cell lines (Roth et al, 2012, Blood). However, the ability of EP to progressively decrease total cellular iron and its relative efficacy in this regard compared with clinically available iron chelators such as Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX) have not been reported. Using a cell based assay system for total cellular iron we therefore compared cellular iron mobilization with EP to that achieved with clinically established iron chelators. METHODS The permanent cardiomyocyte cell line H9C2 derived from embryonic rat ventricle was chosen to model iron mobilization, as heart failure secondary to iron overload is the most common cause of death among patients with transfusion-dependent anaemias. Iron concentration was determined using the ferrozine assay (Riemer et al. Anal Biochem. 2004). A two fold increase of intracellular iron compared to control was obtained by serially treating cells with 10% FBS DMEM media. The cells were then exposed to iron chelators/EP, lysed, and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. The LDH enzymatic viability assay was used to ensure viability was consistently 〉 98% during experiments, and to assess the toxicity of EP on the cardiomyocyte cell line. RESULTS EP induced both dose and time dependent cellular iron removal from cells at 1, 2, 4 and 8 hours. At 1µM, EP was able to remove 42% of total cellular iron following 8 hours of treatment, and 60.1% and 65.62% at 10µM and 30µM respectively (Figure 1). Figure 2 shows that cell viability is compromised at concentrations of 10µM and 30µM to 96% and 92% respectively with eltrombopag, but not with the commercially used iron chelators, DFO, DFP and DFX. However, at 1µM EP the viability of the monolayer is maintained at 〉 98%. The high effects of iron release noted in figure 1 by EP at 10µM and 30µM could be partially attributed to toxicity of the drug on the monolayer. In table 1 the difference in iron removal between EP and commercially used iron chelators after 8 hours of treatment is shown. Interestingly, with EP at only 1µM, 42.9% of cellular iron was removed, compared to 22.7 %, 34.9% and 19.3% in the case of DFO, DFX and DFP respectively, all at higher concentrations of 30µM iron binding equivalents (IBE). DISCUSSION AND CONCLUSION­­ Remarkably low concentrations of EP (1µM) are required to mobilize cellular iron in our cell system while maintaining cell viability at this concentration. This concentration is achievable clinically (Cmax 2-3 µM two to six hours post administration of 75mg orally) (Neito et al, Haematologica, 2011; Deng et al, Drug Metabolism and Disposition, 2011), and when considered alongside with the long plasma half life and elimination in urine and feces, could render it an effective iron chelator for other indications, particularly if combined with existing iron chelation regimes. It would be of interest to explore how EP performed in an animal iron overloaded/MDS model in isolation or as an adjunct to established chelation therapies. Figure 1: Percentage of intracellular iron removed from cardiomyocytes by increasing concentrations of Eltrombopag at different time points up until 8 hours Figure 1:. Percentage of intracellular iron removed from cardiomyocytes by increasing concentrations of Eltrombopag at different time points up until 8 hours Figure 2: Percentage cytotoxicity of cardiomyocyte monolayer following 8 hours of treatment with Eltrombopag and commercially used iron chelators Figure 2:. Percentage cytotoxicity of cardiomyocyte monolayer following 8 hours of treatment with Eltrombopag and commercially used iron chelators Table 1: Comparison of iron mobilization by Eltrombopag and commercially used iron chelators following 8 hours of treatment. Chelator Iron/protein (nmol/mg) SD % iron removal control 26.78 2.5 - DFO 30μM ibe 20.70 2.2 22.70 DFP 30μM ibe 17.43 1.4 34.89 DFX 30μM ibe 21.61 1.0 19.28 Eltrombopag 1μM 15.53 1.9 42.94 Eltrombopag 10μM 10.70 1.2 60.06 Eltrombopag 30μM 9.21 0.3 65.62 Disclosures No relevant conflicts of interest to declare.