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Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez-García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development. First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014). To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18). Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition. Disclosures Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34(+) human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34(+) compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34(+) cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34(+) cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

Background: Flow cytometric immuno-phenotyping of lineage-associated antigens is used in the diagnosis of BCP ALL to distinguish neoplastic B-cells. However, the resultant immunophenotypic expression patterns are inadequate to inform prognosis or choice of therapy. Mass cytometry allows for multi-parametric analysis of single cells to distinguish phenotypic and functional features of subpopulations from leukemia samples. Mass cytometric analysis of pediatric Ph+ BCP ALL constructs a novel model of ALL organized along the normal B cell developmental trajectory (Davis et al., Cell 2014). Leukemic cells share phenotypic features with their normal early B cell counterparts. Critical survival and proliferation signaling is also linked to phenotypic state. Further, the developmental state of leukemic populations common among the patients impacts in vitroresponse to inhibition of BCR-ABL kinase signaling. Methods: Mononuclear cells from diagnostic bone marrow samples were obtained from pediatric patients with Philadelphia chromosome positive BCP-ALL under informed consent (n=21) or healthy controls (n=5). Mass cytometry analysis of 40 proteins was performed at baseline state and perturbed state (IL-7, TSLP, anti-m, dasatinib, tofacitinib, BEZ-235) as previously described (Bendall et al., Science 2011). Analysis was restricted to progenitor and blast populations. Healthy bone marrow samples were gated as previously described along the trajectory of developing B cells (Davis et al., Cell2014). These populations were used as the foundation for a classifier in which each leukemia cell was assigned to its nearest healthy population based on a distance metric (Mahalanobis in nine dimensions). Results: Compared to healthy bone marrow controls, and as expected, ALL samples displayed overexpression of early B cell immunophenotypic markers including CD10 (healthy mean counts 3.83 vs. leukemic 283.3, p=0.03), CD34 (6.26 vs. 80.7; p=0.03), and TdT (2.03 vs.18.9; p=0.002). Leukemic cells expressed lower levels of CD45 and IgM compared to healthy developing B cells. Extended phenotyping revealed conserved patterns of protein expression consistent with different developmental stages in B cell development. We have previously identified the precise developmental ordering of human B cell fractions based on the combined expression of CD34, CD38, CD24, TdT (Davis et al., Cell 2014). ALL samples showed increased numbers of cells occupying B cell progenitor compartments compared to healthy bone marrow controls. To formalize this observation, a single-cell classifier was constructed based on the developmental trajectory of healthy B cells. Each leukemic cell was assigned its most related healthy B cell population based on the expression of nine developmental proteins. Across all samples, the size of the pre-proB (CD34+CD38+TdT+) and proB (CD34+CD38+TdT+CD24+) compartments expanded (12% and 33% in ALL vs. 1% and 2% in healthy, respectively) at the expense of progenitor and preB cell compartments. Interestingly, within a given sample, cells may expand within more than one progenitor compartment such that each leukemia had a corrupted, but distinct B cell developmental trajectory. In two diagnosis-relapse pairs, the relapse sample occupied a more mature phenotype compared to its diagnostic partner. Within the developmental compartments, blast cells retained functional features of their healthy counterparts. Blasts within the ProB cell compartment displayed higher basal levels of pSTAT5, pS6, p4EBP1 and pCreb than blasts in other developmental compartments. These cells were more proliferative based on higher mean expression of Ki67. In healthy bone marrow, cells in this developmental state are characterized by ligand-independent STAT5 activation. Indeed the leukemia’s overall level of pSTAT5 correlated with the percentage of cells in the ProB state (R2=0.71). Similarly, in the leukemic samples, patients with a high percentage of cells in this state were less able to respond to inhibition of STAT5 with the tyrosine kinase inhibitor, Dasatinib than cells in other developmental compartments. Conclusions: Deep proteomic profiling of BCP ALL establishes a single-cell classification linking phenotype with functional attributes of leukemic cells. This data demonstrates that leukemic cells are more or less sensitive to therapeutic intervention based on their developmental state. Disclosures Bendall: Fluidigm: Consultancy. Simonds:Fluidigm: Consultancy, Equity Ownership. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures: Sachs: Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

The classic myeloproliferative neoplasms (MPNs), including primary and secondary myelofibrosis (MF), are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations induce hyperactivated JAK-STAT signaling in cell lines and mouse models. Other mutations have been found recurrently in human MPNs, however, including mutations associated with transformation of chronic MPNs to secondary acute myeloid leukemia (sAML). The aggregate effects of these mutations on in vivo myeloproliferative signaling and disease phenotypes are not yet well understood. While targeted inhibitors of JAK2 have shown activity in MPNs, evidence of a selective effect on the underlying malignant clone has been lacking. These findings suggest that dysregulation of other signaling molecules may be important in MPN pathogenesis, and that therapeutic targeting of these molecules could be beneficial. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed. Mass cytometry is a novel technology that merges flow cytometry with mass spectrometry and enables the simultaneous measurement of 30+ parameters at the single cell level. We utilized this approach to examine bone marrow or peripheral blood samples from four MF patients and five sAML patients, as well as three normal controls. Cells were exposed to five different perturbation conditions ex vivo, including the cytokines thrombopoietin (TPO) and G-CSF, and/or the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 18 surface markers and 16 intracellular signaling effectors and analyzed on a CyTOF mass cytometer. Single cell data was analyzed using traditional gating strategies, as well as with SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets based on shared surface marker expression. These methods grouped cells into distinct cell populations including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict basal and induced activation of each intracellular marker. MF and sAML patient samples exhibited abnormal cytokine-induced signaling that varied in a patient-specific manner. HSPC responses to TPO ranged from hyposensitive to hypersensitive compared with normal. Markedly abnormal basal (unstimulated) signaling in the HSPC compartment was observed in all MF and sAML patients. Each sample exhibited increased total IkBα and/or pCREB, typically accompanied by elevated phosphorylation of one or more additional signaling molecules. Both basal elevation and cytokine hypersensitivity were frequently observed within a MAP kinase signaling axis represented by pERK and downstream targets pCREB and pS6. Conversely, repressed basal STAT1 phosphorylation was observed in all patients. These abnormalities were not exclusive to sAML versus MF, nor specific to patients with or without the JAK2 V617F mutation. Widespread dysregulation of total IkBα, pERK-pCREB-pS6, and pSTAT1 suggest that these signaling effectors may be characteristic of a general myeloproliferative signaling phenotype. Ex vivo ruxolitinib effectively inhibited the majority of observed hypersensitive cytokine-stimulated signaling effects. In contrast, most basal signaling elevations were insensitive to ruxolitinib. The signaling molecules most frequently insensitive to ruxolitinib were those most frequently basally elevated in MF and sAML HSPC, namely pCREB and total IkBα. In contrast, phosphorylation of the JAK2 substrates STAT3 and STAT5 was almost always sensitive to ruxolitinib. These results suggest that targeting of myeloproliferative signaling pathways either downstream or independent of JAK2 activity may be valuable for improved treatment of MF and sAML patients. Ongoing experiments are focused on determining whether constitutive ruxolitinib-insensitive signaling abnormalities can be identified in patients treated with ruxolitinib in vivo, which may underlie the incomplete responses observed clinically. Taken together, these approaches will provide a deeper understanding of altered signaling networks in the context of targeted therapies in MPNs. Disclosures: Oh: Incyte: Consultancy, Research Funding, Speakers Bureau.

Abstract 2341 Background: Growth factors (GFs) that stimulate the proliferation of human hematopoietic stem cells (HSCs) in vitro have been identified, but maintenance of the original stem cell potential has remained a challenge. We have confirmed the observation that UG26–1B6 cells produce factors that enhance the maximum yield in vitro of HSCs achievable with SCF, FLT3-L, IL-3, IL-6, and G-CSF (5 GFs) alone (∼5-fold increase in lympho-myeloid repopulating cells by limiting dilution transplant analysis in NOD/SCID-IL-2Rγc null mice assessed 20 weeks post-transplant). It was therefore of interest to determine whether these different outcomes could be correlated with specific signalling events. Methods: CD34+cell-enriched human cord blood (CB) cells were stimulated with 5 GFs ± UG26 conditioned medium (CM) for defined periods, and then fixed in paraformaldehyde, stained for surface markers, alcohol permeabilized and stained with antibodies specific for multiple intracellular signalling intermediates. Samples were then analyzed using either a CyTOF mass cytometer (34 unique parameters) or a LSR Fortessa cell analyzer (11 parameters). Data analysis was performed using a combination of Cytobank, SPADE, and R using the 'flowCore', 'flowType', 'lattice', 'SamSpectral', and 'gplots' packages. Results: Analysis by CyTOF mass cytometry of 106 CD34+ CB cells after 15 minutes of their stimulation ± 5 GFs ± CM showed that the HSC-enriched subset (Lin−CD34+CD38−CD90+CD49f+cells) exhibited similar responses to the 5 GFs and 5 GFs+CM cocktails (average shifts in median intensity [Δasinh (stimulated-unstimulated)] of 0.32 [-0.14 to 1.96] and 0.37 [-0.02 to 1.78]) with increased phosphorylation of S6, STAT3, STAT5, CREB, IκBa (total protein), AKT, Syk/ZAP70, and ERK1/2. However, the addition of CM did selectively reduce the level of pS6 and pAKT seen with 5 GFs alone, and also caused an increase in pCREB. In addition, activation of pSyk/ZAP70 was detected only in HSCs stimulated with 5 GFs + CM. Phosphoflow analysis of the same subset of cells from a second CB pool confirmed the CyTOF results for pAKT, pSTAT5, pCREB and pERK1/2 (variably) following a 15 minute stimulation but not pSyk/ZAP70. After 2 hours of stimulation, the activation patterns were sustained for most targets for cells exposed to 5 GFs + CM but were reduced in 5 GFs alone - particularly pCREB which was decreased to levels in unstimulated cells. After 24 hours, pAKT and pSTAT5 showed continued activation in response to 5 GFs ± CM, whereas pCREB and pERK1/2 signal were reduced to below starting levels. Hartigans' Dip Test for Unimodality revealed significant deviations from unimodality for the mass cytometry HSC data. Principal component analysis (PCA) of the HSC data followed by subsequent clustering also revealed a number of differentially responsive sub-populations including a broadly cytokine non-responsive, non-apoptotic (cleaved PARP−) population, as well as a minority population with high levels of pSrc and pPLCγ, but reduced activation of other pathways. Further analysis using SPADE revealed decreased cytokine responsiveness with higher CD49f intensity. Weak but significant negative correlations between CD49f and pSTAT3, pCREB, and pS6 and positive correlations with pSrc and pPLCγ (holm-adjusted p values In the CyTOF experiment, multipotent progenitors (MPP; Lin−CD34+CD38−CD90−CD49f– cells) showed broadly similar patterns and levels of activation as HSCs when CM was added (p=0.44), but the median activation was attenuated in 5 GFs alone (p=0.048). MPPs also showed lower levels of multiple phosphorylation events, despite both HSC and MPP populations being Ki67−. This was consistent regardless of cell stimulation status, time point or measurement platform. Conclusions: We have discovered heterogeneity in the signal responses of highly purified fractions of very primitive subsets of human CB cells to culture conditions that affect their self-renewal differentially, that distinguish HSCs from MPPs, and that further subdivide the HSC subset. Among the events surveyed, differences in the timing and intensity of signal activation down common pathways were identified rather than any evidence of selective pathway utilization. These findings hold promise for the identification of how external factors modulate the fates of normal and transformed human HSCs when they are stimulated to divide. Disclosures: No relevant conflicts of interest to declare.

Abstract 1411 Background: Currently available indicators of risk stratification for acute myeloid leukemia (AML) approximate a patient's true prognosis (Rubnitz, J.E., et al., Lancet Oncol, 2010). While signaling networks are central to biological systems they do not lend themselves to easy characterization. As such, network characteristics are not incorporated into risk stratification. There exists a need for computational techniques that enable elucidation of a patient's global signaling state. Flow cytometry is an essential tool for classifying AML surface markers by surface marker expression. Previous work has shown that intracellular signaling responses can be predictive of clinical outcome (Irish et al., Cell 2004; Kornblau et al., Clin. Cancer Res. 2010, Kornblau et al. Blood Cancer J 2011). We hypothesized that there may exist additional layers of information in both cell phenotypes and signaling responses which were not recognized in the low-parameter systems used in these studies, and which, upon characterization, may yield prognostic benefit. Methods: 31-parameter single cell mass cytometry was used to measure the simultaneous co-expression of 16 surface markers and 15 intracellular signaling epitopes in pediatric AML diagnosis bone marrow samples (n=15) and healthy adult bone marrow controls (n=3). Signaling dynamics were measured under 18 stimulation conditions including cytokines and small molecule kinase inhibitors. Single cell resolution enables statistical extraction of correlative relationships which exist among the measured variables. Network inference elucidated the regulatory signaling structure in each patient (Sachs et al. Science 2005, Itani et al. JMLR 2008), and a statistical tool called a decision tree was used to predict the patient's relapse status (Figure 1). Results: The analysis selects network features predictive of clinical outcome, here, relapse status. Of the network edges with a strong statistical signal, the most informative were the relationships between pS6 and pSTAT1, pAKT and cleaved Caspase3, pAMPK and pRb, pRb and pP38, pAMPK and pErk and pCREB and pCbl. Despite the small dataset size, the decision tree correctly classified >85% of the patients, an improvement over a random model which returns 50% accuracy. (The predicted value was excluded from training to avoid “memorization” by the classification model). For a small subset of the patients (3) who relapsed, paired relapse samples were also available. Strikingly, of the network features which consistently differed between the diagnostic sample and its paired relapse counterpart, two overlapped with those found to have predictive information in the above: pAMPK – pRb and pRb – pP38. These results implicate a coordination of metabolism, cell cycle and survival Conclusions: Statistical classification typically relies on large numbers of profiled samples; thus the ability to predict relapse status from diagnostic samples in this small pilot study is a strong indication of the utility of this approach. Predictive accuracy relied on network features and was not achieved using univariate measurements alone. Furthermore, the features extracted by the predictive model determine which correlations help reveal the prognostic fate of a tumor at diagnosis, potentially providing mechanistic insight. These correlative relationships corroborate the roles of metabolism, survival and cell cycle in AML. The application of 31-parameter mass cytometry at the single cell level provided a level of detail that enabled characterization of signaling relationships at high resolution. This proof of principle study will inform future studies of AML signaling networks and prognostic biomarkers. Disclosures: No relevant conflicts of interest to declare.

Abstract 1451 Simultaneously examining multiple epitopes in single cells has become increasingly useful as improvements are made to multi-parametric flow techniques. Increased parameterization has enabled subdivision of functionally distinct cell populations based on an increasing variety of physiological attributes. It has not only helped better define the landscape of “normal” hematopoiesis, but also has been clinically applied in detection of minimal residual disease (MRD) in hematopoietic malignancies. Detection of rare “abnormal” cells is the crux of MRD-based risk stratification where sporadic residual cancer cells are identified through multi-parameter flow cytometry. Current methods for the detection and characterization of cellular populations are generally reliant on manual examination and targeted gating techniques. This approach relies almost entirely on prior knowledge and leaves little room for discovery of novel populations. As the number of parameter per cell increases there is a rising need for dimensionality reduction (DR) methods to resolve high dimensional data “down” into a human-interpretable space. Classical DR, such as principle component analysis (PCA), fail to address the non-linear relationships in cellular phenotypes while newer approaches, such as spanning-tree progression of density normalized events (SPADE), have an inherent level of stochasticity that might adversely affect the robust separation of cellular phenotypes (i.e. discriminating healthy vs. diseased cells). Here, we present a novel algorithm that identifies and characterizes distinct cell populations, preserving the high dimensional information, but providing an interpretable visualization of their phenotypic relationships. This approach was applied to a cohort of normal human bone marrow (BM) specimens to discern a landscape of normal hematopoietic phenotypes. We then contrasted this to overlays of human leukemic bone marrow aspirates (AML and ALL) to understand the extent to which cancer corrupts the shape and form of the landscape. We illustrate the application for automated MRD detection in human leukemia (Figure 1). Method: Our approach, CellSNE, is an adaptation of t-Distributed Stochastic Neighbor Embedding (t-SNE), a non-linear dimensionality reduction algorithm. CellSNE finds a low dimensional mapping of cells that preserves their pairwise distances in a high dimensional space. A distance between each cell to every other cell in the dataset is calculated, based on a vector defined by the combined values of cellular parameters measure. An optimization algorithm then searches for a projection of the points into 2D, in such a way that maximizes the similarity in pairwise distances between the high-dimensional and two dimensional spaces. The resulting 2D projection organizes the sample into subpopulations that conserve the shape and relative distances between each cell. Results/Conclusion: Application of CellSNE to healthy BM clearly separated cells based on their known immune subtype and was confirmed by manual analysis (Figure 1A). The results are robust across data collected from different individuals on different days as well as in analyses conducted using low numbers of single cell parameters, suggesting that healthy BM generally maintains the same cellular population characteristics (or “shape”) across samples. When applied to leukemic BM from patients with AML and ALL CellSNE demonstrates a unique cancer landscape (“shape”) for each patient that is dramatically different from normal (Figure 1B). It is critical to note that despite the overwhelming infiltration by cancer cells, rare “normal” cell populations can still be discerned in the ALL BM. CellSNE succeeded in automatically identifying rare ( Disclosures: No relevant conflicts of interest to declare.

Abstract 703 The classic myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis (PMF) are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations have been shown to cause hyperactivated JAK-STAT signaling in cell lines and mouse models. How accurately these models recapitulate human MPN pathogenesis remains uncertain, as in vivo signaling in MPNs is likely modulated by other genetic changes and regulatory dynamics. In addition, the phenotypic changes that accompany transformation of chronic MPNs to secondary acute myeloid leukemia (sAML) have not been well characterized. While targeted inhibitors of JAK2 have shown activity in MPNs, the incomplete responses observed clinically have called into question the utility of JAK2 as a therapeutic target, suggesting that dysregulation of other signaling pathways may be important in MPN pathogenesis. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed. Mass cytometry is a novel technology that merges aspects of flow cytometry with mass spectrometry – cells are labeled with antibodies conjugated to elemental isotope reporters and then analyzed on the CyTOF mass cytometer. Each mass channel is distinct, such that no compensation is required, thus circumventing the spectral limitations of fluorescence-based flow cytometry and enabling the simultaneous measurement of 30+ parameters at the single cell level. We have utilized this approach to examine multiple signaling effectors in cell populations throughout hematopoietic differentiation. Our initial experiment included samples from three MPN patients (PV, PMF, post-PV sAML), and one normal donor. Cells were exposed to nine different perturbation conditions ex vivo, including cytokines and the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 17 surface markers and 13 dynamic intracellular signaling effectors and analyzed on the CyTOF. Single cell data was uploaded into SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets and creates 2D tree plots based on shared surface marker expression. These plots identified recognizable cell subsets, including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict the relative induction of each intracellular marker in response to each condition. In the HSPC compartment, several expected responses were observed, particularly in PV. Erythropoietin-mediated activation of STAT3 and thrombopoietin (Tpo)-mediated activation of STAT3/5 were enhanced in PV committed progenitors. On a broader level, PV HSPCs exhibited heightened signaling sensitivities involving several cytokines and downstream effectors. Notably, CREB and S6 phosphorylation were strongly induced by Tpo, G-CSF, and IL-3. Ruxolitinib pre-treatment markedly inhibited signaling mediated by Tpo in PV CD34+ cells, indicating that the HSPC compartment can be effectively targeted by ex vivo JAK1/2 inhibition. In contrast to PV, PMF HSPCs exhibited lesser sensitivity to cytokine stimulation. In several instances, such as IL-3 induction of pSTAT5, the responses were in fact suppressed compared to normal. CD34+ HSPC from the sAML patient generally exhibited subnormal signaling responses. However, widespread hyperactivation following exposure to the phosphatase inhibitor pervanadate (PVO4) was observed in sAML CD34+ cells, suggesting that these signaling pathways were activated in vivo, but that feedback inhibition in response to persistent activity led to downregulation of their ex vivo inducibility. Based on these preliminary findings, we hypothesize that the fundamental chronic phase MPN state is one of heightened cytokine signaling sensitivity, while the advanced phase (especially sAML) state is one of tonic or constitutive downstream signaling activity with persistent feedback inhibition on cytokine signaling pathways. To test this hypothesis, experiments with a larger cohort of MPN samples are currently underway. These studies will provide a comprehensive framework of altered signaling in MPNs and provide deeper insights into the role of targeted therapy for MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract 5116 RAS is a known oncogene in AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), however the specific effects of targeting RAS-activated pathways on AML physiology are unclear. NRASG12V transgene repression in an NRASG12V/Mll/AF9 transgenic murine AML model leads to apoptosis and disease remission (Kim et al. Blood 2009). To better understand possibilities for and implications of therapeutic targeting of RAS-activated pathways, we inactivate NRASG12V in this mouse model and characterize the subsequent signaling and transcriptional response. We profiled signaling intermediates and markers of apoptosis and cell cycle using mass cytometry, a next-generation flow cytometry technology, which simultaneously measured the levels of 32 antibody-labeled proteins in single cells. These analyses revealed specific signaling changes that varied with the surface immunophenotype of the AML cells and highlighted relevant RAS-directed signaling pathways. Parallel RNA sequencing and gene expression microarrays revealed that NRASG12V-expressing cells express hematopoietic self-renewal genes and repression of NRASG12V leads to loss of this program. Importantly, the NRASG12V-dependent gene expression program mimics the Mll/AF9-determined, Myb-mediated self-renewal program reported by Zuber et al. (Zuber et al. Genes Dev 2011). These data suggest a novel role for RAS in AML self-renewal capacity by maintaining the Mll/AF9-mediated self-renewal program. Furthermore, inhibition of the PI3K-mTOR pathway decreases viability, in vitro colony formation, and recapitulates the effects of NRASG12V inactivation on self renewal-associated genes, implicating this pathway as the mediator of RAS-directed leukemia self renewal capacity. These data provide rationale for therapeutic targeting of leukemia stem cells via PI3K-mTOR pathway inhibition and for using this mouse model as a tool to test such therapeutic approaches. Since a variety of AML-specific genetic mutations lead to perturbations in RAS signaling, these results may be generalizable to AML with a broader range of mutations. Figure: NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature. Figure:. NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature. Disclosures: Largaespada: Discovery Genomics, Inc. : Consultancy, Equity Ownership; NeoClone Biotechnology, Inc. : Consultancy, Equity Ownership.

Abstract 753 Pediatric B Cell Acute Lymphoblastic Leukemia: Common and Unique Differentiation States Defined by Signaling Response States Background: When mutations or regulatory dysfunction drive inappropriate cell division or survival this sets the stage for cancer initiation or progression. At what point do cells in an early cancer veer from their normal cellular routines to no longer participate in the development of a normal cellular tissue architecture & differentiation heirarchy? Do they still attempt to play out their programming to some degree, or are they “free actors”? The fact that cancers of any given type appear to remain tied to phenotypic classification schemes is illustrated in traditional clinical stratification systems. Paradoxically, cancers are considered (by some) as “heterogeneous”, whereas as a class they appear to recapitulate patterns of clinical responses, gene expression, signaling states. To what extent are these repeated molecular patterns mirrored at the level of differentiation? We mapped, at the single cell level, patterns of expression of markers and phenotypic traits that might be similar or unique across patient subgroups of pediatric B-cell ALL samples using 35 parameter proteomic mass cytometry. Using the features assignable to each single cell, with a statistical reconstruction of most likely similarity of features in 35 dimensional space, we mapped a “common” differentiation tree previously unrecognized by conventional analyses, and demonstrate here how differences in certain markers distinguish, or create, common phenotypic co-expression features across these ALL patients, or suggest patient-specific derailing of differentiation associated with changes in signaling module expression states. Methods: Cryopreserved cells were obtained from 8 pediatric B cell ALL patients under informed consent. 19 surface markers were used to cluster data into metacells of localized similarity displayed in a tree of local relationships via hierarchical cell lineage clustering and spanning-tree progression analysis of density-normalized events (SPADE) (Figure 1) (Bendall et al, Science 6 May2011; 332: 687–696). Results: The blast cell subpopulations comprised the areas of greatest density on the spanning trees–consistent with the fact that these cells are the most prevalent. Blast populations displayed variable expression of B cell precursor surface markers such as CD10, CD19, CD34 and CD38—even though clinical phenotyping placed all patients in a similar class. SPADE analysis detailed unexpected sub-branches prominently observed in certain patients, while absent or weakly represented in others. This confirms common signaling and differentiation states can be observed across patients, but individual patients can manifest unique and prevalent outgrowths of these common malignant differentiation states. Three patients' disease appears to gain a transformative event during a common point at a “pre-B cell development state, leading to local expansion at this halted population of characteristic immunophenotype. Sample ALL04 appears to have an outgrowth of cells with the earliest pre-B cell progenitors, whereas ALL01 is consistent with a maturing pre B cell, gaining expression of CD45. Notably, ALL03, characterized by an MLL rearrangement, clusters with myeloid cells and is CD10 negative, suggesting transformation prior to lymphocyte commitment. The cellular responses to perturbation provides added structure to the cellular subsets; responses to stimulation of the preB cell receptor are present within the majority of blast populations for ALL01 but absent in most for ALL03 and ALL04. We will present this and other findings related to these patients, as well as application of this approach to other tumor types. Conclusion:This high-dimensional immunophenotypic analysis of single cells from primary patient samples reveals an unseen developmental structure within pediatric B precursor acute lymphoblastic leukemia. The developmental stage at which the transformative event occurs informs the characteristic response to perturbation and critically, to drug treatment. *KD and ES contributed equally to this work. Disclosures: Fantl: Nodality, Inc.: Equity Ownership.

Abstract 2565 Acute myeloid leukemia (AML) encompasses a broad spectrum of cellular morphologies. This spectrum exists both within each patient in the form of a malignant developmental hierarchy, and across patients in the form of distinct disease classes with distinguishing mutational or pathological hallmarks. Flow cytometry has long been an essential tool for classifying AML surface markers, and has enabled improved diagnosis, minimal residual disease monitoring, and cell sorting for functional studies. Previous work by our laboratory and others (Irish et al., Cell 2004; Kornblau et al., Clin. Cancer Res. 2010) has shown that clinically relevant heterogeneity also exists at the level of single-cell intracellular signaling capabilities, particularly in response to ex vivo perturbations. Due to the constraints of fluorescent flow cytometry, the limited number of simultaneous parameters measured per cell in these studies was limited (e.g., 3–6). We hypothesized that there may exist additional layers of heterogeneity in both surface marker phenotypes and signaling responses which were invisible to the low-parameter systems used in these studies. To address this challenge, we used a next-generation 31-parameter ‘mass cytometry’ platform to survey the diversity of signaling responses and their relationship to subsets defined by surface marker patterns. We measured the simultaneous co-expression of 16 surface markers and 15 dynamic intracellular signaling epitopes (e.g. phosphorylated kinase substrates) in a cohort of pediatric AML diagnosis bone marrow samples (n=18) and healthy adult bone marrow controls (n=3). Signaling dynamics were measured under 18 stimulation conditions, including a battery of cytokines, chemical stimuli, and small molecule kinase inhibitors. The resulting single-cell data was overlaid and compared using a new, unsupervised flow cytometry analysis and visualization program–SPADE (Spanning-tree Progression Analysis of Density-Normalized Events)–which links rare, transitional, and abundant cell types alike along a branching tree organized by phenotypic progression. Thus, we distilled the diversity of cellular phenotypes, their relative frequencies, and the corresponding signaling dynamics of each cell population onto a single graph structure representing the distinct and overlapping expression patterns in the 16-dimensional “immunophenotypic space” explored by the 21 individuals. Striking signaling and immunophenotypic diversity was observed between the AML samples, particularly in response to pervanadate stimulation. Pervanadate liberates intracellular kinase cascades by broadly inhibiting tyrosine phosphatases, and thus reveals the cell's potential for kinase activity and the interplay of signaling circuits in different cell populations. Interestingly, two transcription factors implicated in AML pathogenesis – CREB and c-Cbl–often exhibited mutually exclusive phosphorylation patterns in different subsets of primitive (CD34+) cells. Specifically, CREB was readily phosphorylated at Ser133 upon pervanadate exposure in the CD34+CD123- cells, while c-Cbl phosphorylation at Tyr700 was restricted to CD34+CD123+ cells. As CD123 (IL-3Ra) has been implicated as a marker of AML stem cells, the finding that these cells are poised for c-Cbl signaling may suggest a potential therapeutic avenue. This work demonstrates the utility of 31-parameter mass cytometry for the simultaneous characterization of multiple cancer stem cell markers and comparison of their expression patterns to the normal tissue hierarchy. These results will inform future studies of AML signaling biology, prognostic biomarkers, and indicators of minimum residual disease. EFS and SCB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Abstract 3160 While oncogene addiction is a well-documented phenomenon, the molecular mechanisms by which oncogene withdrawal triggers cell death are poorly understood. Interrogation of this phenomenon in a manipulatable murine model, coupled to concomitant analysis of human AML samples, could elucidate this phenomenon for therapeutic applications. In order to decipher these molecular mechanisms, we employ a murine model harboring a tetracycline repressible, activated NRAS (NRASG12V) transgene along with an MLL/AF9 transgene to induce AML development. Primary leukemia cells are then transplanted into SCID mice and, upon development of full-blown leukemia, NRASG12V transgene expression is repressed with doxycycline. Previous work has shown that repression of NRASG12V in this model leads to widespread apoptosis of the leukemia cells (Kim et al. Blood 2009). To analyze the kinetics of this response, we found that the tumor burden, as assayed by the white blood cell (WBC) count, declines by 60 hours of doxycycline treatment. NRASG12V message levels are undetectable by 12 hours while protein expression begins to decline after 48 hours. To dissect the signaling network directing the apoptotic response, the phosphorylation status of critical signaling intermediates was analyzed by flow cytometry at time points from 48–96 hours post-doxycycline treatment. This analysis revealed numerous modifications in known NRAS effectors including loss of phosphoErk1/2, phosphoSTAT3, and phosphop38. These alterations correlate with immunophenotypic cell surfaces markers. Whereas leukemia cells expressing mature myeloid markers (Mac1+) do not exhibit alterations in any of the phosphoproteins tested, only Mac1- leukemia cells show meaningful changes in RAS-activated signaling molecules. Whether mouse leukemic stem cells reside in the Mac1+ fraction or in the less differentiated subpopulation remains unclear. These findings suggest that subpopulations of leukemia cells exhibit differential vulnerabilities to oncogene addiction. Furthermore, these studies also reveal that oncogene withdrawal leads to a reduction of the Mac1+Gr1- population and an enrichment of the Mac1-Gr1- and Mac1+Gr1+ populations. Therefore, in addition to effecting apoptosis, oncogene withdrawal leads to alterations in the differentiation status of the leukemia, which could alter the self-renewal capacity of these cells. Using these results, we have designed an extensive antibody panel and are currently using a CyTOF mass spectrometer, a new technology that allows us to perform 30 dimensional measurements to profile immunophenotypic markers and phosphoprotein states with single cell resolution. Simultaneous, high-dimensional single cell profiling of signaling states enables integrative network analysis of these data and as such will allow us to discern pathway dependencies and regulatory relationships that traditional low dimensional flow cytometry cannot (Sachs et al. Science 2005, Sachs et al. IEEE Eng Med Biol Soc 2009). This approach will provide a novel and powerful method to elucidate the critical pathways and leukemic subpopulations that define the response to oncogene withdrawal. Furthermore, these findings will be compared to a concomitant study of 30-dimensional measurements of human AML samples which could facilitate harnessing the oncogene addiction phenomenon in therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

Abstract 2761 Background: High-risk pediatric B precursor lymphoblastic leukemia (preB ALL) remains a serious cause of morbidity and mortality despite therapeutic advances with consequent improvements in survival over the past 30 years. Intracellular protein networks are the functional mediators of differentiation decisions. Perturbing these networks with chemical and biological agents to unveil their signaling potential has shed insight into key networks in other hematologic malignancies such as B cell lymphoma, AML, JMML, and myelofibrosis. The current study was designed to profile evoked signaling responses in NCI high-risk pediatric preB ALL using a next-generation 'mass cytometry' platform (CyTOF.., DVS Sciences). This new technology platform utilizes antibodies conjugated to chelated metal ion tags, and thereby circumvents the limitations of overlapping spectral emission associated with more conventional fluorochrome-conjugated antibodies. Thus, in this preliminary study we measured 13 surface markers and 18 functional intracellular proteins simultaneously at the single-cell level in primary patient samples, with the goal of correlating distinct signaling responses with surface marker expression and response to the Src family tyrosine kinase inhibitor, dasatinib. Methods: All procedures were approved by the Stanford University Human Subject Institutional Review Board. Signaling profiles were measured for cryopreserved diagnostic bone marrow samples from pediatric patients with NCI high-risk pediatric leukemia in the basal state and in response to a 15 minute treatment to 18 different extracellular modulators, with or without prior exposure to dasatinib for 30 minutes. An example is shown in Figure 1. Cell populations were gated based on surface marker expression. The activation states of 18 functional intracellular markers were measured within the leukemic blasts (Figure 1). Results: Within the bulk blast population, distinct signaling responses could be discerned correlating with surface marker heterogeneity. Blasts were manually gated into 5 distinct populations based on surface expression of CD45 and CD10. These populations demonstrated further heterogeneous expression of surface markers CD34, CD38 and CD19 as well as heterogeneous activation states of intracellular proteins such as pERK, NFkB, and pStat5. Robust activation of phospho-proteins proximal to the preB cell receptor was observed following treatment with preBCR cross-linking antibodies or with tyrosine phosphatase inhibitors. This robust signaling was abrogated by pre-treatment of leukemic blasts with dasatinib. Unexpectedly, concurrent treatment with IL-7 or preB cell receptor crosslinking antibodies and dasatinib sensitized cells to dasatinib inhibition, resulting in a synergistic suppression of phosphorylation below baseline values (Figure 2). Discussion: Together, these results suggest that intracellular signaling and response to therapeutic agents such as kinase inhibitors may differ based on the complex interaction of the leukemic cell and its microenvironment. This preliminary study shows the feasibility of using a novel mass cytometry platform to simultaneously monitor 18 intracellular signaling mediators on a per cell basis in primary samples from pediatric high risk ALL patients to a level of detail not previously possible. This approach allowed the identification of cells with a robust signaling phenotype, based on the activation of pERK, pStat5 and NFkB, members of pathways known to be involved proliferation and survival. These data identify potential targets for therapeutic agents such as the tyrosine kinase inhibitor dasatinib. inhibited preBCR signal transduction, and synergy with other biological agents. Further studies are ongoing to measure multidimensional signaling with CyTOF technology in additional high risk pediatric ALL samples with the goal of identifying and guiding the most beneficial therapeutic choices. EFS and KLD contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.