David B. Rosen

Dynamic Single-Cell Network Profiles in Acute Myelogenous Leukemia Are Associated with Patient Response to Standard Induction Therapy

Purpose: Complete response to induction chemotherapy is observed in ∼60% of patients with newly diagnosed non-M3 acute myelogenous leukemia (AML). However, no methods exist to predict with high accuracy at the individual patient level the response to standard AML induction therapy. Experimental Design: We applied single-cell network profiling (SCNP) using flow cytometry, a tool that allows a comprehensive functional assessment of intracellular signaling pathways in heterogeneous tissues, to two training cohorts of AML samples (n = 34 and 88) to predict the likelihood of response to induction chemotherapy. Results: In the first study, univariate analysis identified multiple signaling “nodes” (readouts of modulated intracellular signaling proteins) that correlated with response (i.e., AUCROC ≥ 0.66; P ≤ 0.05) at a level greater than age. After accounting for age, similar findings were observed in the second study. For patients <60 years old, complete response was associated with the presence of intact apoptotic pathways. In patients ≥60 years old, nonresponse was associated with FLT3 ligand–mediated increase in phosphorylated Akt and phosphorylated extracellular signal-regulated kinase. Results were independent of cytogenetics, FLT3 mutational status, and diagnosis of secondary AML. Conclusions: These data emphasize the value of performing quantitative SCNP under modulated conditions as a basis for the development of tests highly predictive for response to induction chemotherapy. SCNP provides information distinct from other known prognostic factors such as age, secondary AML, cytogenetics, and molecular alterations and is potentially combinable with the latter to improve clinical decision making. Independent validation studies are warranted. Clin Cancer Res; 16(14); 3721–33. ©2010 AACR.

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

Background Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative. Methodology/Principal Findings Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management. Conclusions/Significance These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.

Functional pathway analysis in acute myeloid leukemia using single cell network profiling assay: Effect of specimen source (bone marrow or peripheral blood) on assay readouts

Single cell network profiling (SCNP) is used to simultaneously measure the effects of modulators on signaling networks at the single cell level. SCNP‐based biomarker assays predictive of response to induction therapy and relapse risk in acute myeloid leukemia (AML) patients are being developed. Such assays have typically used bone marrow (BM) as the sample source of blasts. Because circulating peripheral blasts are detectable in ∼65% of AML patients and peripheral blood (PB) sampling is less invasive than BM sampling, this study was performed to assess the effect of sample source on AML blasts signaling as measured in SCNP assay.

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

Background Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways. Methodology and Principal Findings Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct “DNA damage response (DDR)/apoptosis” profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the “DDR/apoptosis” proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents. Conclusions and Significance Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.

AKT Signaling as a Novel Factor Associated with In Vitro Resistance of Human AML to Gemtuzumab Ozogamicin

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ1 derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ1 -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ1 and, by extrapolation, other DNA damage-based therapeutics.

Development and validation of a single‐cell network profiling assay‐based classifier to predict response to induction therapy in paediatric patients with de novo acute myeloid leukaemia: a report from the Children's Oncology Group

Single cell network profiling (SCNP) is a multi‐parameter flow cytometry technique for simultaneous interrogation of intracellular signalling pathways. Diagnostic paediatric acute myeloid leukaemia (AML) bone marrow samples were used to develop a classifier for response to induction therapy in 53 samples and validated in an independent set of 68 samples. The area under the curve of a receiver operating characteristic curve (AUCROC) was calculated to be 0·85 in the training set and after exclusion of induction deaths, the AUCROC of the classifier was 0·70 (P = 0·02) and 0·67 (P = 0·04) in the validation set when induction deaths (intent to treat) were included. The highest predictive accuracy was noted in the cytogenetic intermediate risk patients (AUCROC 0·88, P = 0·002), a subgroup that lacks prognostic/predictive biomarkers for induction response. Only white blood cell count and cytogenetic risk were associated with response to induction therapy in the validation set. After controlling for these variables, the SCNP classifier score was associated with complete remission (P = 0·017), indicating that the classifier provides information independent of other clinical variables that were jointly associated with response. This is the first validation of an SCNP classifier to predict response to induction chemotherapy. Herein we demonstrate the usefulness of quantitative SCNP under modulated conditions to provide independent information on AML disease biology and induction response.

Assessing signaling pathways associated with in vitro resistance to cytotoxic agents in AML

This study uses single cell network profiling (SCNP) to characterize biological pathways associated with in vitro resistance or sensitivity to chemotherapeutics commonly used in acute myeloid leukemia (AML) (i.e. cytarabine/daunorubicin, gemtuzumab ozogamicin (GO), decitabine, azacitidine, clofarabine). Simultaneous measurements at the single cell level of changes in DNA damage, apoptosis and signaling pathway responses in AML blasts incubated in vitro with the above drugs showed distinct profiles for each sample and mechanistically different profiles between distinct classes of agents. Studies are ongoing to assess the clinical predictive value of these findings.

Systems biology analysis of immune signaling in peripheral blood mononuclear cells (PBMC) of melanoma patients receiving ipilimumab; basis for response biomarker identification.

Systems biology analysis of immune signaling in peripheral blood mononuclear cells (PBMC) of melanoma patients receiving ipilimumab; basis for response biomarker identification Drew Hotson, Ryan Alvarado, Andy Conroy, Santosh Putta, Ester Simeone, Assunta Esposito, Mariaelena Capone, Gabriele Madonna, Antonio M Grimaldi, David B Rosen, Spencer Liang, Alessandra Cesano, Carmela Cacciapuoti, Paolo A Ascierto, Rachael E Hawtin

Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP)

BackgroundHomologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity.MethodsFlow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates.ResultsEtoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples.ConclusionsSCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.