ALBERT

Description: Abstract 1263 Poster Board I-285 Background In conjunction with antigen-driven responses, ligand-independent signaling (termed tonic signaling) through both the pre-B cell receptor and B-cell receptor has an important role in B cell development, maturation and survival. In addition to the recognized role of CD79 alpha and CD79 beta BCR signaling, tyrosine phosphatases can impact tonic BCR signaling (Wienands et al. PNAS, 93 p.7865 (1996), Monroe Nat. Rev. Immunol. 6 p.283 (2006)). We previously subjected chronic lymphocytic leukemia (CLL) cells with modulators of BCR signaling and monitored their responses using flow cytometry-based Single Cell Network Profiling (SCNP). Of the many signaling modulators studied, hydrogen peroxide treatment (a general inhibitor of tyrosine phosphatase activity) augmented BCR signaling in a subset of CLL patient samples evaluated. In the remaining samples there was an apparent lack of response to hydrogen peroxide. These data suggested that differential phosphatase activity proximal to BCR signaling was driving the biology of these two patient groups. Objectives Studies were designed to evaluate whether there were any associations between tonic and/or ligand-dependent BCR signaling and in vitro sensitivity to fludarabine, as well as whether such response profiles showed a relationship to the hydrogen peroxide-dependent signaling we observed previously. Methods 23 CLL samples and 7 healthy PBMCs were treated with anti-m alone, hydrogen peroxide alone or the combination for 10 minutes. Separate aliquots of the same sample were exposed to F-ara-A for 48 hours. SCNP was carried out on gated B cells with quantitation of single cell measures of intracellular phosphorylated kinases and adaptor proteins downstream of the BCR. Additionally, the relative activation status of several protein markers of the apoptotic cascade (cytoplasmic cytochrome C, cleaved caspase 3, and cleaved PARP) was measured. Results As previously observed, CLL samples could be segregated into one of two groups exhibiting either responsive or refractory signaling after exposure to hydrogen peroxide alone. Moreover, responsive signaling in CLL cells was correlated in that all the measured components of the canonical B cell receptor network (p-Lyn, p-Syk, p-BLNK, p-PLC-gamma-2, p-Erk and p-Akt) showed the same phosphorylation response: either augmented in unison, or not activated at all. In vitro F-ara-A treatment (48 hours in the presence of 1mM F-ara-A) of parallel samples from these same CLL patients identified distinct populations of apoptosis responsive and refractory cells. Surprisingly, the capacity of patient samples to show augmented BCR signaling in response to hydrogen peroxide was associated prominently with the ability of cells in these patients to exhibit apoptotic proficiency to F-ara-A in vitro. This implies a link between mechanisms governing apoptosis in these CLL cells, survival pathways, and cell states that govern the role of phosphatase activity and BCR signaling potential. Conclusions This study reveals a link between tonic BCR signaling and regulation of apoptosis pathways. This suggests that the subgroup of CLL patients with active phosphatase activity (which suppresses BCR responses) have cell populations that are responsive to F-ara-A, a standard drug in CLL therapy. Conversely, the presence of CLL cells in a patient sample that remain unresponsive to hydrogen peroxide repression of phosphatase activity appear to identify patient samples which cannot undergo apoptosis in response to in vitro F-Ara-A exposure. The clinical implications of this work will be the focus of future translational studies. Disclosures Evensen: Nodality Inc.: Employment, Equity Ownership. Palazzo:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership.

Description: Abstract 2745 Poster Board II-721 Background: Gemtuzumab Ozogamicin (GO, Mylotarg), a humanized CD33 monoclonal antibody linked to calicheamicin was approved by the US FDA for use as a monotherapy in patients older than 60 years with relapsed acute myeloid leukemia (AML) unfit to tolerate standard salvage therapy. GO is internalized rapidly after infusion, and calicheamicin, a potent enediyene, is subsequently released and acts as a cytotoxic agent by causing double strand DNA breaks. Currently GO is in multiple clinical trials as a single agent or in combination with other therapies for both induction and consolidation treatment of various clinical subgroups of AML. However, the mechanisms of action and resistance of GO are incompletely understood and it is unclear which patient subgroups benefit from GO-based therapy. Single cell network profiling (SCNP) has shown promise as a methodology wherein multiple signaling networks are measured after treatment with an exogenous modulator such as a growth factor, cytokine or therapeutic agent and the identified signaling profiles can be used as clinical and therapeutic enablement tools. Objectives: SCNP using multiparameter flow cytometry was used to identify intracellular pathways that were associated with responsiveness or refractoriness to in vitro GO exposure in both cancer cell lines and primary AML samples. Methods: Signaling pathways emphasizing DNA damage response, cell cycle, apoptosis and drug transporter activity were measured by SCNP after in vitro exposure of cell lines and AML primary samples to clinically relevant concentrations of GO. Samples were processed for cytometry by paraformaldehyde /methanol fixation and permeabilzation followed by incubation with fluorochrome-conjugated antibody cocktails that recognize cell surface proteins to delineate cell subsets and intracellular signaling molecules. Results: In cell lines, responsiveness to in vitro GO exposure was defined as a) induction of DNA Damage as measured by increased p-ATM, p-Chk2 and p-H2AX, b) cell cycle arrest at G2/M as measured by increased cyclin B1 and DNA content & c) induction of apoptosis as measured by cleaved PARP and viability dyes. Of note, inhibition of drug transporter activity in 2 MDR-1+ cell lines did not restore GO responsiveness, suggesting the presence of additional relevant resistance mechanisms in these cell lines. In primary AML diagnostic samples, DNA damage and apoptosis pathway readouts were able to identify responsiveness or refractoriness to GO exposure. In the GO responsive profile, induction of both DNA damage responses and apoptosis were seen. Within the refractory samples, two distinct profiles were observed: a) robust and early induction of DNA damage response without apoptosis and 2) delayed and attenuated DNA damage response without apoptosis. Conclusions: Characterization of intracellular Cell Cycle, DNA Damage, and Apoptosis networks in single cells after GO exposure distinguishes GO responsive from refractory AML cells. Further, these pathway signatures provide information about mechanisms of refractoriness. (e.g. a block between a successful DNA damage response and initiation of apoptosis versus a block in the initial induction of DNA damage after GO exposure). The ability of the same profiles to predict clinical responses to the drug will be tested in future studies. Disclosures: Rosen: Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Soper:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality Inc.: Employment, Equity Ownership.

Description: Abstract 3827 Poster Board III-763 Background ON 01910.Na, a small molecule multikinase inhibitor, promotes G2/M arrest and apoptosis. Key targets for this inhibitor include Plk1 (polo-like kinase, a cell cycle regulator), Cdk1, (cyclin dependent kinase, a mitotic regulator) and the PI-3 kinase pathway (Ramana Reddy et al. J. Med. Chem. 2008, Park et al, Oncogene, 2007, Gumireddy et al., Cancer Cell, 2005). The drug has been shown to have anti-tumor activity in in vitro and in vivo models. Phase I studies in 〉100 advanced cancer patients revealed that the drug is well tolerated. Further, in several ongoing Phase 1 clinical trials in patients with myelodysplastic syndromes (MDS), positive effects on hematological indicators have been noted (Sloand et al, ASH 2008). Based on these data, a Phase 2 single-arm study is in progress to assess the efficacy and safety of the drug in IPSS Intermediate-2 and High risk MDS patients. Single Cell Network Profiling (SCNP) using flow cytometry is a platform that measures multiple fluorescent parameters (up to 10) in each cell, including both surface markers and intracellular signaling proteins in response to extracellular network inputs. By simultaneously measuring the effects of drug exposure on several pathways within each cell type in a heterogeneous patient tissue sample, valuable data can be gained about drug interactions with specific cellular pathways and cell type selectivity. This information has potential implications for dose/schedule optimization and development of patient stratification biomarkers. Objectives Studies were designed to evaluate the in vitro effects of ON 01910.Na, at clinically relevant concentrations, on intracellular pathways in the human GM-CSF-dependent erythroblastic TF-1 cell line using SCNP in order to monitor transitional changes in the cell cycle, with a focus on the G2-M phase and to perform dose-dependent titrations of drug using these cell cycle readouts. Methods The reagents chosen to measure cell cycle readouts were fluorochrome-conjugated antibodies that recognize cyclin B1, p-histone H3(S28) and p-Cdk1(Y15) and 4'6'-diamino-2-phenylindole (DAPI), a fluorescent dye that binds strongly to DNA. The phosphorylation status of p-histone H3(S28) and p-Cdk1(Y15), and the level of cyclin B1 expression are all determinants of the G2-M and/or M phase of the cell cycle. Dose dependent titrations of ON 01910.Na and its inactive analog ON 01911 were performed over a dose range starting at 10-5 M and decreasing to 10-10 M (dose range which includes pharmacologically achievable concentrations in humans) with 3-fold serial dilutions for eleven points after an exposure to the drug for either 24 or 48 hrs. Cells were processed for multiparameter flow cytometry by fixation, permeabilization and incubation with fluorchrome-conjugated antibodies. Results The data showed that at 24 hours after ON 01910.Na exposure there was a simultaneous increase in phosphorylation of histone H3(S28), a decrease in phosphorylation of Cdk-1(Y15), and accumulation of cyclin B1. These data suggest that ON 01910 exposure disrupted the G2/M cell cycle transition leading to mitotic arrest with subsequent apoptosis. TF-1 cell DNA content measured by DAPI verified this to be the case as increases in G2/M and sub-G1 (a measure of apoptotic cell death) were simultaneously observed. No significant effects on G2/M targets were observed when TF-1 cells were exposed to ON 01911, indicating the effects of ON 01910.Na on the cell cycle were specific to the drug. Maximal effects of ON-01910.Na on cell cycle signaling molecules were observed at a drug concentration of 0.37 mM and no further changes were seen at higher concentrations. These effects were also observed at 48 hours, although with more cell death. Conclusions These data indicate that intracellular phosphorylation changes of histone H3(S28) and Cdk-1(Y15), in addition to accumulation of cyclin B1 with subsequent apoptosis, reflect possible mechanisms of action of ON 01910.Na. The assay will be used in ongoing clinical trials to measure the pharmacodynamic activity of the drug in MDS patient samples pre- and post-treatment. Disclosures: Soper: Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Wilhelm:Onconova Therapeutics Inc: Employment. Cosenza:Onconova Therapeutics Inc.: Consultancy. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Cesano:Nodality Inc.: Employment, Equity Ownership. Greenberg:Nodality Inc.: Research Funding; Onconova Therapeutics Inc.: Research Funding. Fantl:Nodality, Inc.: Employment, Equity Ownership.

Description: Signals propagated through the B cell receptor (BCR) guide the maturation and survival of B cells and might factor in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). BCR signaling in CLL cells was investigated at the single-cell level using multiparametric flow cytometry. Concurrent analysis was performed using fluorochrome-conjugated antibodies specific for B-cell surface antigens and a panel of antibodies recognizing specific phospho-peptide epitopes within a selected group of intracellular signaling proteins. CLL samples from patients (N=6) showed weak or minimal signaling activity at p72SYK/p70ZAP, Erk1/2, B-Cell linker protein (BLNK) and phospholipase-Cγ-2, (PLCγ2) when stimulated only at the BCR with anti-μ crosslinking, whereas a robust signal was observed in a control Ramos B cell line. The low-level signaling in CLL cells could be accounted for by either a defect in activation of a key protein required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP-1, SHIP-2, SHP-1, or SHP-2. To determine whether phosphatases were preventing or dampening BCR activation in CLL samples, CLL cells were treated with hydrogen peroxide (H2O2), a physiologic phosphatase inhibitor generated during BCR signaling that has been used previously to reveal dysregulated BCR signaling in follicular lymphoma (Sing et al., Cell 2005, Reth., Nat. Immunol. 2002, Irish et al., Blood 2006). H2O2 treatment of CLL cells that had molecular features associated with indolent disease (e.g. use of mutated immunoglobulin heavy chain variable region genes (IgVH) and a low level expression of ZAP70) induced high-levels of phosphorylated p72SYK/p70ZAP, ERK1/2, BLNK, and PLCγ2, independent of surface F(ab)2anti-μ ligation. In contrast, CLL-B cells that had molecular features associated with more aggressive disease (expression of unmutated IgVH and high-level expression of ZAP-70) were significantly less responsive to this treatment, even in the presence of F(ab)2anti-μ. Exposure of blood B cells from healthy donors to H2O2 failed to elicit a substantial increase in phosphorylation of these same intracellular signaling proteins. These studies reveal a previously unrecognized, constitutive high-level phosphatase activity in CLL cells that express mutated IgVH and low levels of ZAP-70, possibly contributing to the attenuated signaling observed in these cells following surface IgM ligation. Because this high level of constitutive phosphatase activity was not observed in CLL cells that express unmutated IgVH with high-level expression of ZAP70, flow cytometric measurement of the increased phosphorylation triggered by H2O2 could provide for a useful means with which to distinguish leukemia cells from patients with indolent versus aggressive CLL. Evaluation of additional samples is ongoing to test this hypothesis.

Description: Abstract 2788 Background: Normal hematopoiesis changes with age through unknown mechanisms. Low risk (LR) myelodysplasia (MDS) is characterized by cytopenias arising through inefficient hematopoiesis. We hypothesized that both of these differences might result from changes in responsiveness to external signaling. To test this hypothesis, we utilized SCNP, a multiparametric flow cytometry-based assay that simultaneously measures both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators, quantitatively at the single cell level (Kornblau et al. Clin Cancer Res 2010). Methods/Objective: SCNP was applied to examine baseline and intracellular signaling responses induced by the extracellular modulators EPO and GCSF in bone marrow (BM) mononuclear cells (BMMC) derived from healthy donors (n=15) and MDS (n=9) patients. The effects of donor age on signaling profiles in healthy BMMC were compared between samples collected by BM aspirate from 6 subjects aged 23–43 years (“younger”) and from the BM present in hip replacement samples from 9 subjects aged 54–82 years (“older”). Signaling profiles were also determined for 9 LR MDS patients aged 53–83 years and compared to the age-matched healthy “older” control. Metrics used for analysis included fold change, total phosphorylation levels, and the Mann-Whitney U statistic model. Results: There were no differences in the frequency of CD34+ cells (R2= 0.006, p= 0.78) between “younger” and “older” healthy donor samples, suggesting any differences observed in signaling would likely be due to donor age rather than sample source. There was no age-related difference in functional signaling ability in response to GCSF-induced p-STAT1, p-STAT3, & p-STAT5 levels; however, early erythroblasts and normoblasts from healthy “older” donors were significantly less responsive to EPO, as measured by induced phospho (p)-STAT5 levels than those derived from healthy “younger” donors (e.g. R2= 0.654 p=0.008 for erythroblasts and R2=0.628 p=0.0004 for normoblasts). Signaling profiles classified Refractory Anemia with Excess Blasts (RAEB) patients into 2 categories based on differences in EPO- and GCSF-induced signaling (Table 1). Compared to age-matched healthy “older” controls, one subset was characterized by a high % of RBC precursors (CD45lo nRBC) and increased p-STAT5 levels in response to EPO and the other subset by a high % of myeloid cells with robust GCSF-induced p-STAT3 & p-STAT5 responses in both total myeloid and CD34+ cells. By contrast, patient samples with Refractory Anemia with Ringed Sideroblasts (RARS) had a high % of CD45lo nRBC but lacked robust p-STAT5-induced signaling after modulation with EPO. Conclusions: Overall, these data show the feasibility of using the SCNP assay in BM samples to functionally characterize signaling pathways simultaneously in different cell subsets of healthy donors and patients with MDS. In healthy individuals, age-related differences in EPO signaling were discovered. In LR MDS, differences in signaling were observed between cases and in comparison to the data from healthy controls. Deciphering signaling profiles in healthy donor versus MDS patient samples may result in improved, biologically-based disease classification that informs more effective patient management. The clinical relevance of these findings in terms of disease course and treatment is currently under investigation. Disclosures: Cleary Cohen: Nodality Inc.: Consultancy, Equity Ownership. Huang:Nodality Inc.: Consultancy, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.

Description: Abstract 4906 Background: The mechanism of action (MOA) of AZA and DAC is considered to be predominantly via DNMT inhibition. However these drugs are also cytotoxic as nucleoside analogs, and AZA inhibits RNA translation and protein synthesis. Both are clinically efficacious in HR MDS but patient responses differ. We hypothesized that the relative contributions of these functions to overall MOA may be at the basis of this clinical divergence. Previous studies by others (Hollenbach et al, PLOS One 5(2) e9001) in AML cell lines demonstrated that both drugs reduced DNMT1 levels at clinically relevant concentrations and induced apoptosis. However, they differed in effects on the cell cycle: AZA was active across cell cycle phases while DAC induced a G2-M shift. Here we apply single cell network profiling (SCNP) to analyze the effect of AZA or DAC on cell cycle and DNMT levels in bone marrow mononuclear cell (BMMC) specific subsets (CD34+, nRBC, lymphocytes, myeloid cells) from primary HR MDS and healthy donors (HEA). Methods: SCNP is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and changes in intracellular proteins in response to extracellular modulators (including drugs). SCNP was used to measure cell cycle (DAPI stain), apoptosis (amine aqua and c-Parp) and levels of DNMT1, DNMT3a and DNMT3b at baseline and after treatment for 24 hours with AZA (2. 5uM) and DAC (0. 625uM) in AML cell lines (U937, TF-1), primary BMMC from HEA 〉 60 years of age (n=15) and HR MDS (n=10, RAEB, int-2/high) patients. The Mann-Whitney U statistic was used for analysis. Results: Cell cycle and apoptosis. Treatment of U937 cells with AZA increased the subG1 cell fraction (i. e. apoptotic cells) from 0 to 12% and decreased the percentage of cells in S phase from 37% to 24%. Conversely treatment with DAC increased the fraction of cells in S phase (from 37% to 54%), consistent with DAC intercalation into DNA, with no significant effects on the subG1 fraction. In TF-1 cells, only AZA significantly induced apoptosis as measured by both amine aqua (10%) and c-Parp (5%). Similar results were observed in primary HR MDS samples: in 4 of 8 HR MDS samples AZA treatment resulted in apoptosis in CD34+ cells (median= 20. 5% induction) while DAC did not affect CD34+ cell viability. DNMTs levels. In U937 cells AZA decreased all 3 DNMTs levels (DNMT1(41%), DNMT3a(26%), DNMT3b(40%)), more effectively than DAC (4%, 4% and 2% decrease, respectively). In primary samples, nRBC subsets from HR MDS showed higher basal levels of DNMT3a and DNMT3b compared to healthy age matched BMMC (p= 0. 003 and p=0. 029 respectively). In the latter, AZA and DAC both decreased the levels of DNMT1, DNMT3a and DNMT3b in all BMMC cell subsets (lymphocytes, nRBC, myeloid and CD34+). The effects of these drugs in HR MDS contrast with healthy samples, as summarized in Table 1. Specifically: 1) both drugs increased the levels of all three DNMTs in lymphocytes, 2) DNMT1 levels were increased by both drugs in lymphocytes, myeloid and CD34+cell subsets, and 3) effects of the two drugs diverged with respect to effects on DNMT 3a and 3b in myeloid cell subsets (Table 1). Conclusions: In vitro treatment for 24 hrs with clinically relevant concentrations of AZA or DAC decreased baseline DNMTs levels in cell lines and healthy BMMC, consistent with their role as promoters of genomic hypomethylation. However, these drugs have strikingly different effects on primary HR MDS BMMC cell subsets. While MDS is not typically considered a disorder of lymphocytes, DNMTs levels increased in response to either AZA or DAC suggesting an alteration in HR MDS lymphocytes vs. HEA lymphocytes. The combined differential activities of AZA and DAC on cell cycle, intracellular DNMTs levels and apoptosis in specific cell subsets (in particular DNMT3a levels in myeloid and CD34+ HR MDS BMMC and absence of DAC-induced apoptosis) suggest the inhibition of post-transcriptional processing by AZA contributes significantly to cytotoxicity and may explain differences in clinical activities of the 2 drugs. Disclosures: Cohen: Nodality, Inc.: Consultancy. Huang:Nodality, Inc.: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership.