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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 employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V). We performed gene expression microarray and RNA sequencing of our AML cells in the presence and absence of NRASG12V. By employing computational approaches to explore NRASG12V-responsive genes in our model, we found that NRASG12V 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. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival. We used CyTOF (mass cytometry) for a multiplexed analysis of RAS-dependent signaling intermediates, and found that Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. Using RAS-pathway inhibitors, we found NRASG12V 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. Recent work has shown that NRASG12V has bimodal effects in hematopoietic stem cells (Li et al. Nature 2013). To understand the mechanism of these bimodal effects, we have performed single cell RNA sequencing on our AML model. We expect that these analyses will reveal the cell-type specific NRASG12V –mediated mechanisms of leukemia self renewal. Citation Format: Zohar Sachs, Rebecca S. LaRue, Hanh T. Nguyen, Karen Sachs, Nurul Azyan Mohd Hassan, Ernesto Diaz-Flores, Susan K. Rathe, Aaron L. Sarver, Sean C. Bendall, Ngoc A. Ha, Miechaleen D. Diers, Garry P. Nolan, Kevin M. Shannon, David A. Largaespada. NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B15. doi: 10.1158/1557-3125.RASONC14-B15

Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells

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.

It is now well appreciated that intra-tumor heterogeneity is of critical importance. There is remarkable molecular variability between and within populations of tumor cells, driven by both genetic and epigenetic variation. We address the challenge of identifying and characterizing tumor sub-populations through a combined experimental and computational approach. In two examples, we will demonstrate approaches to address each type of heterogeneity. Seventy percent of melanoma tumors have activated, and dependent on, MAPK pathway. However, the phenotypic response to MAPK inhibition is heterogeneous, both in vitro and in patients. While this heterogeneity is well characterized, we attempted to better understand its underlying mechanism. We used gene expression data both before and after pathway inhibition in a panel of cell lines to decipher the network structure under MAPK, and to identify network rewiring that contribute to the phenotypic response. To our surprise, the targets of the MAPK pathway vary dramatically between cell lines. We found that most targets are context-specific, regulated by MAPK only in a subset of samples. We therefore developed an algorithm to detect context-specific targets, and we used those targets to identify the pathways that are being regulated by MAPK. We found that NFkB and STAT3 activation status is correlated with apoptosis levels induced by MAPK inhibition. Furthermore, we show that they protect cells from the cytotoxic effects of MAPK inhibition. We employ mass cytometry, which accurately measures the expression and phosphorylation states of more than forty proteins in thousands of single cells, including surface proteins and signaling molecules. We present results on both healthy bone marrow and bone marrow from both ALL and AML patients. To understand abnormal, we first built a more accurate map of normal, modeling B-cell development in the marrow at unprecedented resolution. We measured 8 healthy marrows with 42 antibody parameters with mass cytometry targeting a multitude of phenotypic markers, intracellular signaling molecules, hallmarks of cell cycle and apoptosis all in the context of in vitro perturbations relevant to B cell development (including IL-7 and BCR crosslinking). We developed a graph based trajectory algorithm (wanderlust) that can trace a continuous progression from the hematopoietic stem cells, through the progenitor cells, to the final, committed B-cells. Our derived map of healthy B-cell development revealed the order and timing of developmental events at unprecedented resolution. The resulting multidimensional data was modeled using wanderlust and the predicted trajectory was then used to inform a traditional ‘gating’ analysis of the data and provide a higher resolution view of human B cell development than previously published. This healthy map was used to help provide a backbone on which to further understand how these trajectories are dysregulated in ALL. Citation Format: El-ad David Amir, Oren Litvin, Jacob Levine, Sean C. Bendall, Kara L. Davis, Erin F. Simonds, Tanya Schild, Mark Rocco, Neal Rosen, Garry P. Nolan, Dana Pe'er. Towards rationale therapy: Dealing with intertumor and intratumor heterogeneity. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA30.

Acute myeloid leukemia (AML) can present with multiple concurrent subclones at diagnosis. Subclone-specific mutations may confer resistance to molecular-targeted drugs through loss of antigen expression or rewiring of intracellular signaling pathways, leading to relapse. Deep sequencing approaches improve the detection of rare subclones, but the ability to prospectively identify subclones (i.e. without relapse material) is limited, and the effects of subclone-specific mutations on phenotype are poorly understood. In the course of a broader study of oligoclonal pediatric AML patients, we focused investigation on a diagnosis bone marrow sample from a patient harboring 3 presumed subclones (two with distinct NRAS-G12D, NRAS-G13D mutations) as determined by whole-genome sequencing (WGS). We employed a combination of 31-parameter mass cytometry, deep sequencing, FACS sorting, and computational modeling to produce a detailed profile of the subclonal genotypes and phenotypes in this patient. The 3 anticipated subclones did not correlate with a clear subset of surface markers in the mass cytometry analysis. Instead, we observed a single continuous ‘differentiation trajectory’ from progenitor-like to monocyte-like blasts. To dissect this trajectory, 7 distinct myeloid/progenitor subsets were FACS-sorted, plus T/B cells as a non-leukemic control. For each subset we performed capture-based deep sequencing of 307 tumor-specific variants (Tier 1: 13; Tier 2: 33; Tier 3: 261; median depth: 1420x). Shifts in allele frequencies among the FACS-sorted subsets provided critical information to a Bayesian mixture modeling algorithm, allowing identification of 5 subclones, as opposed to the 3 subclones anticipated by WGS. The inferred subclonal genotypes were validated and further refined by targeted single-cell Sanger sequencing of multiple Tier 3 loci. Although all 5 subclones were present throughout the differentiation trajectory, some were enriched in certain phenotypic states or ‘reservoirs’. For example, the NRAS-G12D subclone was enriched in a progenitor-like subset, but its daughter subclone, which harbored an additional mutation in RAC2 (implicated in HSC engraftment), was enriched in the more differentiated subsets, suggesting an opposing effect. Notably, the T/B cell population harbored 2 tumor-specific Tier 1 mutations at >15% allele frequency, suggesting its presence in a preleukemic, multilineage-competent HSC. Taken together, subclone-specific mutations appear to skew cells toward either progenitor or mature phenotypes, but the developmental trajectory enforced by parental mutations is resistant to change. Furthermore, characterization of subclones is possible at diagnosis, and may improve the selection of targeted therapies. *Contributed equally: EFS, SCB & ALG Citation Format: Erin F. Simonds, Sean C. Bendall, Amanda L. Gedman, Jacob H. Levine, Kara L. Davis, Harris G. Fienberg, Astraea Jager, El-ad D. Amir, Ina Radtke, Wendy J. Fantl, Dana Pe'er, James R. Downing, Garry P. Nolan. Deep sequencing of immunophenotypically distinct subsets in acute myeloid leukemia reveals reservoirs of genetically distinct subclones along a conserved differentiation trajectory. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2013-3178

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.

SummaryTissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and “corrupted” developmental processes including cancer.