Abstract
BackgroundSingle 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 FindingsThirty 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 SignificanceCharacterizing 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.
Highlights
Proteomic technologies that can monitor aberrant cell signaling in disease hold promise in enabling more accurate diagnosis and prognosis, as well as predicting response to therapeutic agents [1,2,3]
Patient Samples Thirty four diagnostic cryopreserved non-M3 Acute Myeloid Leukemia (AML) peripheral blood mononuclear cell (PBMC) samples taken from patients within the University Health Network, University of Toronto were used in this study
Given the prosurvival role of Jak/Stat and PI3K signaling in cancer, functional performance testing via Single cell network profiling (SCNP) analysis of these pathways was carried out in AML blasts after their exposure to a panel of modulators known to play a role in myeloid biology through engaging these pathways [24,25,35,36,37,38]
Summary
Proteomic technologies that can monitor aberrant cell signaling in disease hold promise in enabling more accurate diagnosis and prognosis, as well as predicting response to therapeutic agents [1,2,3]. Single cell network profiling (SCNP) utilizing flow cytometry differs from most proteomic technologies by measuring modulated phospho-protein and other signaling protein responses at the single cell level [3,4]. The net result of these molecular changes is alteration of proteins within signal transduction networks that drive functional changes in cell proliferation, survival, differentiation, progression and cellular responses to drug therapy [16,17,18,19,20,21]. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways
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