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TO THE EDITOR: BCR-ABL -negative myeloproliferative neoplasms (MPNs) are a group of clonal stem cell disorders characterized by the overproduction of mature myeloid cells. However, MPN patients also have a 2.8- to 3.4-fold increased risk of developing a lymphoproliferative disorder (LPD) compared

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents & Royalties.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the selfrenewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Therapy resistance and relapse in acute myeloid leukemia (AML) are driven by leukemia stem cells (LSCs). Recent evidence highlighting functional and genetic heterogeneity among LSC subclones underscores the importance of capturing the entire LSC compartment in studies of LSC biology. Although LSCs are often enriched in the CD34+CD38- cell fraction, they are frequently detected in other phenotypic fractions, and in some cases are restricted to the CD34- population. In order to discover novel LSC markers, we examined genes differentially expressed between functionally-validated LSC+ and LSC- cell fractions obtained from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs. CD200 expression in 57 primary AML samples was analyzed by flow cytometry using anti-human CD200 clone 1B9(kindly provided by Trillium Therapeutics Inc.). CD200 was present on a greater proportion of CD45dim blasts compared to more differentiated CD45high non-blast populations (54.4% versus 21.7%, p To validate CD200 as a LSC marker, leukemic blasts were sorted from 15 primary AML samples based on CD45 and CD200 expression and transplanted into NSG mice. Samples were selected based on either the presence of both CD200+ and CD200- blasts, or CD200 expression on 5% on blasts. Overall, these results indicate that CD200 expression can be used to segregate LSCs from bulk leukemia cells. CD200 expression may be a particularly useful LSC marker in cytogenetically normal AMLs with NPM1 mutation (CN-AMLNPM1c), which have low or negative CD34 expression and commonly possess CD34- LSCs. Among 20 CN-AMLNPM1c samples, the proportion of CD200+ blasts was higher than that of CD34+ blasts irrespective of FLT3-ITD status, although there was a trend towards higher CD200 expression in FLT3-ITD+ samples. In xenotransplantation assays, 7 of 8 CN-AMLNPM1c samples tested contained CD200+ LSCs while the remaining sample contained both CD200+ and CD200- LSCs. Principal component analysis of gene expression profiles demonstrated that functionally-validated CD200+ LSC-containing fractions from CN-AMLNPM1c patients clustered separately from LSC fractions from NPM1wt or cytogenetically-abnormal cases, and were enriched for stem cell genes by gene set enrichment analysis. Furthermore, ATAC-Seq analysis demonstrated greater chromatin accessibility in CD200+ LSC-containing versus CD200‒ LSC-depleted fractions from CN-AMLNPM1c patients, with unique enrichment of HOX motifs. These data validate CD200 as an LSC marker in CN-AMLNPM1c cases. In summary, CD200 is a valuable tool for capturing heterogeneous LSC populations including both CD34+ and CD34- LSCs in many primary AML samples. It will be particularly useful for future studies of LSCs in CN-AMLNPM1c where CD34 expression does not identify LSCs. Disclosures No relevant conflicts of interest to declare.

Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring. Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. Disclosures Mullighan: Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space. To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism. RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients. Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.

Background Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), the most common form of ALL in adults, is a highly aggressive blood malignancy defined by the BCR-ABL1 fusion. Although inhibitors targeting the BCR-ABL1 oncoprotein, such as imatinib, have significantly improved clinical response rates for this disease, a subset of patients are refractory to therapy or respond initially but relapse soon after. ABL1 kinase domain mutations partly explain differential responses in patients; however, for the majority of cases, a molecular basis that can reconcile this clinical observation is lacking. Methods Flow-sorted blasts from 53 primary samples, 49 de novo Ph+ ALL and 4 lymphoid blast crisis CML, were subjected to RNA sequencing (RNA-seq) and whole genome sequencing (WGS). Response rates were tracked using BCR-ABL1 transcript levels from patient blood. Results Hierarchical clustering of transcriptome data produced two molecular subgroups of Ph+ ALL. One subgroup, which we termed 'Core-B', upregulated key regulators of B-lymphoid differentiation including IL7R and MS4A1 (CD20). By contrast, the second subgroup upregulated an expression program related to hematopoietic stem cell (HSC) and myeloid differentiation, with upregulation of KIT, CD34, MPO, CSF3R, and GATA3. We termed this subgroup 'Aberrant-Stem-Myeloid' (ASM). These subgroups displayed a striking disparity in response rates to intensive chemotherapy with imatinib. Whereas 'Core-B' patients showed highly durable responses often lasting many years, 'ASM' patients frequently relapsed (4% vs 43% relapse; p=0.007). We used WGS analysis to investigate the genetic basis of these molecular subtypes. 'Core-B' Ph+ ALLs were enriched for deletions in PAX5, a B-cell specification gene, and CDKN2A/B, tumor suppressors. The 'ASM' subtype lacked these genetic alterations; instead, these leukemias were enriched for deletions in EBF1, an early B-cell lineage factor that represses T-lymphoid and myeloid lineages and is expressed before PAX5 in B-cell lineage differentiation. Accordingly, blasts from 'ASM' leukemias with EBF1 deletions showed decreased CD19 antigen expression and upregulation of myeloid antigens by clinical flow cytometry. Rare cases with concurrent EBF1 and PAX5 deletions showed expression features of both 'ASM' and 'Core-B' leukemias. Mutations observed in myeloid leukemias (TET2, RUNX1) were only present in the 'ASM' subtype. Loss of IKZF1, found in 77% of cases, also displayed distinct patterns between the two subgroups; deletions leading to the dominant negative isoform (Ik6) were enriched in the 'Core-B' subgroup (45% vs. 14%; p=0.019) while monosomy 7 and large deletions encompassing IKZF1 were enriched in the 'ASM' leukemias (41% vs. 10%; p=0.017). In 1 of 4 diagnosis/relapse patients analyzed, a molecular switch from 'Core-B' at diagnosis to 'ASM' at relapse was observed. The diagnostic 'Core-B' clone from this patient harbored a PAX5 mutation that was lost at relapse, whereas the relapsed 'ASM' clone harbored trisomy 21 and a RUNX1 mutation. Altogether, our data suggest that the 'ASM' leukemias emerge through dysregulation of genes earlier in lympho-myeloid specification compared to 'Core-B' leukemias. These findings led us to investigate if the 'ASM' subtype originates from an HSC and the 'Core-B' subtype originates from a B-cell progenitor. We first looked at the distribution of the long (p210) and short (p190) isoforms of BCR-ABL1 in the two subtypes. The p210 isoform, also the hallmark of CML, is speculated to arise in an HSC, and the p190 is thought to arise in a B-cell progenitor. Neither the p190 or p210 BCR-ABL1 isoform was enriched in either subgroup. We resolved highly purified HSC and progenitor subsets from CD34+CD19- cells, functionally evaluated by methylcellulose assays, and subjected them to a sensitive nested-PCR strategy. Cases from both the 'ASM' and 'Core-B' subtypes showed HSC/myeloid progenitor involvement regardless of the BCR-ABL1 isoform. This data suggest that the cell-of-origin does not play a role in establishing the molecular subtype of leukemia blasts. Conclusion There are two distinct molecular subtypes of Ph+ ALL that demonstrate differential responses to treatment and emerge from independent mutational routes. Moreover, the key genetic determinants that form the molecular subtype are secondary driver alterations that lie downstream of BCR-ABL1. Disclosures No relevant conflicts of interest to declare.

Adjusting hematopoietic hierarchy In adults, more than 300 billion blood cells are replenished daily. This output arises from a cellular hierarchy where stem cells differentiate into a series of multilineage progenitors, culminating in unilineage progenitors that generate over 10 different mature blood cell types. Notta et al. mapped the lineage potential of nearly 3000 single cells from 33 different cell populations of stem and progenitor cells from fetal liver, cord blood, and adult bone marrow (see the Perspective by Cabezas-Wallscheid and Trumpp). Prenatally, stem cell and progenitor populations were multilineage with few unilineage progenitors. In adults, multilineage cell potential was only seen in stem cell populations. Science , this issue p. 10.1126/science.aab2116 ; see also p. 126

SummaryAlterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance. Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.

Acute myeloid leukemia (AML) is a hierarchical disease in which the bulk blast population is sustained by a minority population of leukemia stem cells (LSC). Evidence of functional heterogeneity in the LSC compartment, including variable responses to chemotherapy, underscores the importance of examining the entire stem cell compartment in studies of LSC biology. However, there are currently no phenotypic markers that can consistently segregate LSCs within the leukemic blast population. Although LSC activity is most often enriched in the CD34+CD38- cell fraction, LSCs are also frequently detected in other phenotypic fractions, and in some cases are absent from CD34+ fractions. Thus, LSC studies that focus only on CD34+CD38- blasts may miss biologically important clones that are present in other phenotypic cell fractions. To identify novel markers that will enable better enrichment of LSC activity, we examined gene expression data obtained from functionally validated LSC+ and LSC- cell fractions sorted from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs. In normal adult bone marrow and cord blood samples, CD200 is expressed on >95% of CD34+CD38- cells, and expression decreases on CD34+38+ cells, suggesting that CD200, similar to CD34, is a stem cell marker. Flow cytometric analysis of AML patient samples (n=57) demonstrated that CD200 expression was present on a greater proportion of CD45dim blasts compared to CD45high non-blast populations (69.2% vs 4.5%, p To test whether CD200 can segregate LSC activity within the CD45dim blast population, we sorted 14 primary AML samples into cell fractions based on CD45 and CD200 expression followed by transplantation into cohorts of NSG mice. AML samples were prescreened for leukemic engraftment ability and were selected for sorting if: 1) CD45dim blasts comprised both CD200+ and CD200- cells or 2) the CD200+ fraction was High CD200 expression was significantly associated with shorter overall survival in univariate analysis in multiple independent AML cohorts, but was not significant in multivariate analysis due to association with NPM1 mutation. NPM1-mutated samples exhibited low CD200 expression on the bulk cells. Interestingly, 7 of the 9 AML patients with CD200+ LSCs had normal karyotype and NPM1 mutation. Our results demonstrate that a CD200-based sorting strategy can successfully enrich and/or segregate LSC activity, and separate LSCs from preL-HSCs in primary AML patient samples. This will now enable direct functional studies of the biological properties of these related but distinct stem cell populations. Furthermore, CD200 will be a valuable tool for the study of LSCs in the subset of NPM1-mutated AML. Disclosures No relevant conflicts of interest to declare.

Background: The hematopoietic roadmap is a compilation of the various lineage differentiation routes that a stem cell takes to make blood. On several occasions over the last six decades, the murine roadmap has been reconceived due to new information overturning old dogmas. The human roadmap, which describes the extraordinary throughput of more than three hundred billion cells daily has changed little, with the classical model of hematopoiesis still prevailing. In this model, blood differentiation initiated at the level of stem cells must pass through a series of increasingly lineage-restricted oligopotent and then unipotent progenitor intermediates. As cellular differentiation flows from stem cells to unipotent cells, the oligopotent progenitors are the quintessential step where lineage choices are consolidated. One such critical oligopotent intermediate is the common myeloid progenitor (CMP), believed to be the origin of all myeloid (My), erythroid (Er) and megakaryocyte (Mk) cells. While murine studies are beginning to challenge the existence of oligopotent progenitors, a comprehensive analysis of human myelo-erythroid differentiation is lacking. Moreover, whether the pool of oligopotent intermediates is fixed across human development is unknown. The classical roadmap is the only guide that describes human hematopoiesis and consequently our view of blood homeostasis and disease is intimately tied to this conception. Rationale: The differentiation roadmap taken by human hematopoietic stem cells (HSCs) is fundamental to our understanding of blood homeostasis, hematopoietic malignancies and regenerative medicine. Results: We mapped the cellular origins of My, Er and Mk lineages across three timepoints in human blood development: fetal liver (FL), neonatal cord blood (CB) and adult bone marrow (BM). Using a new cell-sorting scheme based on markers linked to Er and Mk lineage specification (CD71 and BAH1), we found that previously described populations of multipotent progenitors (MPPs), CMPs, and megakaryocyte-erythroid progenitors (MEPs) were considerably heterogeneous and could be further purified beyond current definitions. Nearly 3000 single cells from 11 cellular subsets from the CD34+ compartment of FL, CB and BM (33 subsets in total) were evaluated for their My, Er and Mk lineage potential using an optimized single-cell assay. In FL, the ratio of cells with multilineage versus unilineage potential remained constant in both the stem cell (CD34+CD38-) and progenitor cell (CD34+CD38+) enriched compartments. By contrast in BM, nearly all multipotent cells were restricted to the stem cell compartment, whereas unilineage progenitors dominated the progenitor cell compartment. Oligopotent progenitors were only a negligible component of the human blood hierarchy of adult BM leading to the conclusion that multipotent cells differentiate into unipotent cells directly. Mk-Er activity predominately arose in the stem cell compartment at all developmental timepoints. In CB and BM, most Mks emerged as part of mixed clones from HSCs/MPPs, indicating that Mks directly branch from a multipotent cell and not from oligopotent progenitors like CMP. In FL, a striking 80% of single-cell clones with Mk activity were derived from a novel progenitor in the stem cell compartment, although less potent Mk progenitors were also present in the progenitor compartment. Interestingly, in a hematological condition of HSC loss (aplastic anemia), Mk-Er but not My progenitors were more severely depleted, pinpointing a close physiological connection between HSC and the Mk-Er lineage. Conclusion: Our data indicate that there are distinct roadmaps of blood differentiation across human development. Prenatally, Mk-Er lineage branching occurs throughout the cellular hierarchy. By adulthood, both Mk-Er activity and multipotency are restricted to the stem cell compartment, whereas the progenitor compartment is composed of unilineage progenitors forming a 'two-tier' system, with few intervening oligopotent intermediates. Disclosures No relevant conflicts of interest to declare.

Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL. Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing. Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples. We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2). Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases. We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway. Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.

Recent studies have shown that several miRNA are differentially expressed in hematopoietic stem cells (HSC) and involved in regulating self-renewal, pointing to a new axis of epigenetic control of HSC function. Murine studies have documented a role for miR-125a in regulating HSC as miR-125a enforced expression augments self-renewal. We examined whether these attributes are evolutionarily conserved within human hematopoiesis. Lentiviral vectors over-expressing miR-125a (miR-125OE) were developed and HSC function was investigated using xenotransplantation of CD34+ CD38- human umbilical cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). miR-125OE resulted in significantly increased human bone marrow (BM) chimerism at 12 and 24 weeks post-transplantation and splenomegaly. Within enlarged spleens, there were significantly increased proportions of CD34+CD19+CD10+CD20-B lymphoid cells suggesting a partial B cell differentiation block at the pro-B cell stage. In the BM, CD41+ megakaryocytes, GlyA+ erythroid and CD3+ T cell populations were significantly expanded. Within the primitive compartment, multi-lymphoid progenitors (MLP) were massively expanded by 12 weeks, followed by a combined reduction of immuno-phenotypic HSC and multi-potent progenitors (MPP) by 24 weeks. Given this loss of immuno-phenotypic HSC, we wondered whether stem cell function was compromised in vivo. Secondary transplantation with limiting dilution (LDA) revealed that stem cell frequencies were increased by 4.5 fold in miR-125OE recipients. Using lentivirus sponge-mediated inhibition of miR-125 (miR-125KD) in CD34+CD38-human CB, we were able to directly link these effects to miR-125: B cells increased at the expense of T cells; immuno-phenotypic HSC increased with a concomitant loss of MLP; and functional HSC were decreased by 2.5 fold using secondary LDA assays. Together, these data strongly suggest that miR-125a expression levels regulate human HSC self-renewal and lineage commitment. Since HSC frequency increased so substantially upon miR-125OE, we asked whether more committed cell populations might also be endowed with enhanced self-renewal. Highly purified populations of HSC, MPP and MLP and CD34+CD38+ committed progenitors were transduced and transplanted cells into xenografts. Unexpectedly, miR-125OE transduced CD34+CD38+ progenitors produced a substantial graft after 12 weeks. Control transduced CD34+CD38+ cells did not engraft and only control transduced HSC generated a disseminating graft in recipient mice. miR-125OE transduced HSC and MPP generated robust engraftment, while MLP did not. In all cases, xenografts generated by CD34+CD38+ and MPP transduced with miR-125OE showed multi-lineage repopulation. Moreover, the miR-125OE grafts from CD34+CD38+ and MPP recipients were durable as secondary transplantation generated multi-lineage grafts for at least 20 weeks in 5/7 and 6/10 recipients, respectively; no control transduced groups generated secondary grafts. Thus, the enhancement of self-renewal by enforced expression of miR-125a occurs not only in HSC, but also in MPP and to an as yet unidentified subpopulation within the CD34+38+ committed progenitor compartment. Using protein mass spectrometry, we identified and validated a miR-125a target network in CD34+ CB that normally functions to restrain self-renewal in more committed progenitors. Together, our data suggest that increased miR-125a expression can endow an HSC-like program upon a selected set of non-self-renewing hematopoietic progenitors. Our findings offer the innovative potential to use MPP with enhanced self-renewal to augment limited sources of HSC to improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

The multistep pathogenesis of Down Syndrome (DS)-associated pre-leukemia and subsequent progression to acute leukemia is one of the better characterized of all human blood malignancies. Children with DS have a 150 fold increased risk of developing acute megakaryoblastic leukemia (AMKL) and greater than 30 fold increased risk of developing B cell acute lymphoblastic leukemia (B-ALL). DS-AMKL is often preceded in late fetal development or soon after birth by a pre-leukemic syndrome termed transient myeloproliferative disorder (TMD), which is characterized by high numbers of abnormal megakaryocytes and megakaryoblasts in the circulation, spleen and liver. Previous work has demonstrated that constitutional trisomy 21 results in expansion of megakaryocyte-erythroid progenitors (MEP) in fetal liver (FL) with a concomitant reduction in fetal pre-pro-B cells. The expanded MEP population subsequently acquires an N-terminal truncating mutation in the transcription factor GATA1 (termed GATA1s), leading to selective expansion of a pre-leukemic erythromegakaryocytic blast population. While the majority of DS-TMD cases spontaneously resolve within 3 months, up to 15% of DS-TMD neonates can develop lethal progressive liver fibrosis. Progression to AMKL following spontaneous resolution of TMD is associated with acquisition of at least one additional germline mutation. While murine models implicate a role for trisomy 21 and GATA1s in the leukemogenic process, they do not faithfully recapitulate the pathology of the human disease. Previous attempts to model DS-associated TMD through xenotransplantation of DS-FL and DS-TMD cells have proven technically challenging. Therefore, there remains a need for a human model to investigate the genetic steps required for initiation of DS-TMD and progression to DS-AMKL. We previously identified a leukemia stem cell (LSC)-associated miRNA signature by sorting 13 adult AML patient samples into 4 sub-populations based on CD34/CD38 expression, followed by supervised analysis guided by the in vivo leukemia initiating capacity of each sub-population in an optimized xenotransplant model. Interestingly, the top three LSC-associated miRNA candidates are all located on chromosome 21. To determine the role of these miRNA in human leukemogenesis, we engineered a tri-cistronic lentivector for enforced expression. Compared to control vector-transduced cells, tri-cistronic vector-transduced Lin‒CD34+CD38‒ cord blood (CB) cells generated a myeloproliferative syndrome in xenotransplanted mice, with splenomegaly, enhanced CD45+ human bone marrow cellularity and blocked B cell development at the pro B cell stage. Human grafts were enriched for CD45+CD33+CD117+CD123+CD41lo/CD42lo cells in bone marrow, peripheral blood, spleen and liver. In the CD45‒ compartment, a distinct lineage switch was observed, with CD41+ megakaryocytic output supplanting normal CD235+ erythroid output. High numbers of CD41+CD42b+CD61+CD34lo human platelets were detected in peripheral blood and spleen. Blood films revealed large dysplastic platelets and megakaryoblast-like cells. Histology showed hCD45+ packed bone marrow cavities, with loss of normal architecture. Bone marrow, spleen and liver all showed extensive reticulin deposition. In the lineage negative (Lin-) fraction of BM, we observed an expansion in the proportion of human MEP and multi-lymphoid progenitors (MLP). To further model leukemic progression, we expressed GATA1s in combination with our tri-cistronic miRNA vector. Mice transplanted with double transduced cells showed intermediate levels of splenomegaly and bone marrow cellularity compared to mice transplanted with cells transduced with tri-cistronic vector alone. The addition of GATA1s induced a complete loss of B cell development while restoring erythroid development. In human Lin‒ cells isolated from the BM, addition of mutant GATA1s further augmented the proportion and total numbers of MEP while restoring the MLP compartment to normal levels. These data demonstrate that we have generated a human xenograft model of DS-TMD through enforced expression in normal CB cells of a tri-cistron comprising 3 LSC-associated miRNA in combination with mutant GATA1s. With this model in place, we plan to further interrogate the genetic lesions involved in progression from DS-TMD to DS-AMKL. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with a relapse rate of up to 80% depending on patient age and AML subtype. AML is organized as a functional cellular hierarchy and is sustained by a rare population of leukemia stem cells (LSC). Recent work suggests that LSC properties influence therapy response, overall survival, and disease relapse. In order to develop more effective novel therapies that target this rare cell population; it is imperative that we better understand LSCs at the molecular level. Although it is generally accepted that oncogenic mutations underlie cancer initiation and progression, most studies have focused on protein coding genes. However, there is increasing recognition that non-coding RNAs can also play a role in leukemogenesis. microRNAs (miRNA) are a family of small non-coding RNAs that function as important regulators of mRNA stability and translation of protein-coding genes with significant roles in maintenance of human hematopoietic stem cells (HSC) (Lechman et. al., Cell Stem Cell, 2012). To understand the functional role of miRNA in human hematopoiesis, we generated HSC- and leukemia stem cell (LSC)-specific microRNA (miRNA) profiles by microarray analysis of sorted cell fractions from umbilical cord blood (CB) and AML patient samples that have been validated in xenograft assays. We identified ten miRNA candidates over-represented in HSC and/or LSC. To determine whether these were functional and impacted on stem cell properties we transduced lineage depleted CB cells with lentivirus expressing either a candidate miRNA or control vector followed by transplantation into immune deficient mice. Three miRNAs (miR-125b, miR-130a, miR-155) conferred a competitive growth advantage while four miRNAs (miR-99a, miR133a, miR194, miR-196b) conferred a growth disadvantage. miR-125b, a top LSC array candidate, showed the most pronounced phenotype with an overt expansion of transduced cells (19% to 96.2%) and enlarged spleens (2.4 fold increase). Detailed flow cytometric analysis of the miR-125b human grafts in recipient mice revealed a greatly expanded proportion of multi-lymphoid progenitors (MLP), in comparison to HSC and multi-potent progenitors. Furthermore, upon enforced in vivo expression of miR-125b in three AML patient samples, we observed large increases in the primitive primitive CD34+CD117+ populations (CD34+: 2.4-4.6 fold increase; CD117+: 1.3-4.1 fold increase) and a decrease in the proportion of differentiated CD14+/CD15+ cells (CD14+: 6.2-7.6 fold decrease; CD15+: 1.2-6 fold decrease) in leukemic grafts. Limiting dilution assays into secondary recipients revealed up to a 34-fold increase in LSC frequency compared to control vector transduced AML cells. Overall, these data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal. miR-125b belongs to an evolutionarily conserved family consisting of three paralogs (miR-125a; miR-125b1; miR-125b2). Recent studies present strong evidence for a role of the miR-125 family in normal and malignant murine hematopoiesis, yet comprehensive functional inconsistencies remain in regards to the precise roles for each paralog. We are currently carrying out additional enforced expression studies directly comparing these family members in vitro and in vivo in order to clarify the functional roles of miR-125a (a top HSC array candidate) and miR-125b (a top LSC array candidate) in both normal and malignant human hematopoiesis. These studies will determine whether the miR-125 family is a suitable target for therapy of hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2370 Aplastic anemia (AA) encompasses a spectrum of marrow failure disorders that include paroxysmal nocturnal hematuria (PNH), myelodysplastic syndrom (MDS) and acute myeloid leukemia (AML). Despite advances in therapy, approximately a tenth of patients will evolve to severe MDS or AML. A major unresolved question is the nature of the initiating cell that eventually expands during the aplastic phase and gives rise to secondary disease. A critical first step to approach this problem is to characterize the primitive stem and progenitor compartment in AA, taking advantage of the recent advances in phenotypic profiling of primitive human hematopoiesis (Doulatov et al Cell Stem Cell 2012). To this end, we established a 12-parameter flow-sorting scheme for deep phenotypic characterization of the CD34 compartment in AA patients that we used to quantify the gains and losses of all major cellular entities during the aplastic phase. These studies represent the first comprehensive analysis of AA. In 7 out of 10 patients, the proportion of mature myeloid, B cells and NK cells was reduced by greater than 5 fold, whereas the percentage of T cells remained indistinguishable against controls. Within the CD34+CD38-primitive progenitor compartment, the relative number of CD38-CD90+CD45RA- hematopoietic stem cells (HSC) and multipotent progenitors (MPP) reduced. The CD34+CD38+ progenitor populations including common myeloid progenitors (CMP) and megakaryocyte erythroid progenitors (MEP) were either dramatically reduced (>5 fold) or virtually undetectable. Although we hypothesized that absence of phenotypic HSC would result in the absence of its immediate downstream progeny, this was not the case in most of patients. Within the CD34+CD38+ progenitor compartment, MEPs were the most affected population compare to CMPs or GMPs in more than 80% of cases. Interestingly we noticed in 8 of 12 patients, the proportion of granulocyte macrophage progenitors (GMP), defined by FLT3 and CD45RA expression, was unperturbed. To validate whether GMPs from AA patients were functional, we measured the in vitro colony forming capacity of GMP sorted populations. Clonal analyses of these cells in methylcellulose culture showed that these cells have similar potentials and cloning efficiency as normal donor cells. Cloning frequency, size of the clones and number of clones generated in methylcellulose and morphology was also comparable between GMPs from patients and normal donors. We then asked whether these GMPs are the result of a clonal expansion using mitochondrial DNA (MtDNA) analysis. This assay assesses the mutation rate of the D loop region of MtDNA from clones derived from single GMP cells from a patient or healthy donor to gain insight into the diversity within the clones. Our preliminary data suggest that the aplastic anemia GMPs share a closer ancestry compare to GMPs from healthy donor. The in depth quantification of the CD34+ compartment in AA patients that our study provides has directly established that phenotypically defined HSC are profoundly reduced, and this loss has a subsequent impact on downstream components of the hematopoietic hierarchy. The impact of HSC depletion is not a universal loss of progenitors, some progenitors can become overrepresented. Despite the small scale of this study, the overrepresentation of GMP versus other myeloid progenitors in patients with limited clonal heterogeneity suggests that GMP may be a potential candidate for the initiating cell that eventually evolves to MDS or AML. Disclosures: No relevant conflicts of interest to declare.

SummaryThe hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.

Despite high survival rates for children with acute lymphoblastic leukemia (ALL), only 40% of adult patients will achieve long-term disease-free survival, and relapses in both pediatric and adult ALL are often fatal. Most current therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells, yet recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. The functional properties and clinical relevance of these neoplastic subclones remain undefined. Genome wide copy number analysis of matched diagnostic and relapse ALL samples identified that in 50% of patients, the clones present at relapse are not the dominant clones at diagnosis, but have evolved from an ancestral pre-leukemic clone (Mullighan et al., 2008). In order to investigate the functional consequences of clonal evolution in disease progression and therapy resistance, we performed limiting dilution analysis of 3 diagnostic and 14 paired diagnostic/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics, by transplantation into immune-deficient mice (xenografts). In one patient, the leukemia-initiating cell (LIC) frequency was 7.65 fold higher in the relapse sample than at diagnosis, while another patient showed the reverse with a 5.81 fold higher LIC frequency in the diagnostic sample. Two patients showed no significant differences in LIC frequency from diagnosis to relapse. LIC frequency varied from 1 in 14.2 to 1 in 4802 CD19+ blast cells. Interestingly, in 50% of the paired patient samples, transplantation of cells from the relapse sample gave rise to greater leukemic dissemination to the spleen and/or central nervous system of recipient mice in comparison to the diagnostic sample, despite similar levels of engraftment in the bone marrow. This data suggests that although the LIC frequency in B-ALL remains high and relatively static between diagnosis and relapse, relapse cells acquire increased invasive properties. To investigate the clonal composition of 3 diagnostic B-ALL samples, we undertook copy number variation (CNV) analysis of xenografts generated at both limiting and high transplanted cell doses. In all 3 samples, we detected subclones in the xenografts that were distinct from the predominant clone in the primary patient sample. We performed network analysis on these subclones and identified differentially enriched pathways, including differential expression of anti-apoptotic and apoptosis regulation pathways, providing evidence of putative functional differences. These results support the existence of functionally diverse subclones within diagnostic samples as well as functional diversity between the subclones present at diagnosis and relapse. Ongoing in depth genomic analysis of the diagnosis/relapse paired samples will add to our understanding of the functional role of the subclones identified at diagnosis in the establishment of disease relapse. In summary, these experiments will provide further insight into the functional heterogeneity present in B-ALL and the drivers of lymphoid leukemogenesis that lead to therapy failure and disease relapse. Disclosures: Danska: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.