Single cell network profiling by flow cytometry as a platform to reveal pathway signatures and drug response profiles for use in personalized medicine

Purpose: With the aim of developing a platform for the rapid biological characterization of patient-specific tumors, which could assist in making rational therapeutic decisions, we combined signaling pathway analysis and drug response profiling across multiple blood cell populations from a single patient with chronic myelomonocytic leukemia (CMML) using single cell network profiling (SCNP) by flow cytometry.

Experimental Design and Procedures: A PBMC sample from an 83-year-old man with CMML was divided into multiple experimental arms to 1) measure basal and cytokine (SCF, G-CSF, EPO, TPO, IL-27, Flt3L, IL-3) induced signaling to the JAK/STAT, MAPK, and PI3K pathways in multiple cells subsets; 2) measure the inhibition of evoked signaling by small molecule inhibitors targeting PI3K, mTOR, Mek, JAKs, and Flt3; and 3) simultaneously measure apoptosis and cytostasis in multiple cell subsets in response to 47 treatments in vitro, including approved (n=11) and investigational (n=9) drugs, dosed as single agents or in combination after a 48-hour compound exposure. All results were obtained within 1 week of receipt of sample.

Results: Signaling Arm. Profound differences in cytokine signaling were observed in four defined myeloid cell populations as well as when compared with healthy cells from a control PBMC sample, which included the following:

• Immature blasts (CD34+CD11b-) had robust signaling to known survival and proliferation factors, including IL-27, G-CSF, SCF, and Flt3L. Kinase inhibitors were identified that blocked downstream signaling.

• Intermediate blasts (CD34-CD11b-) signaled in response to IL-27, a result not seen in healthy PBMCs.

• cKit+ leukemic cells had constitutive activation of the mTOR pathway, which was not inhibited by rapamycin. Apoptosis/Cytostasis Arm. SCNP allowed for multi-endpoint, multisubpopulation, analysis of drug sensitivity and allowed treatments to be rank ordered using 1 of 3 endpoints: 1) inhibition of the fraction of cells in M-Phase, 2) inhibition of the fraction of cells in late S/G2 phase, or 3) induction of apoptosis. Findings included:

• An 8-fold greater response was observed for decitabine or fludarabine in the c-Kit- versus the c-Kit+ population (M-phase readout).

• In both myeloid populations, Aurora kinase inhibitors (SNS-314 and VX-680) completely blocked cells in M-phase and were non-toxic to lymphocytes.

• Kinase inhibitors were significantly more effective when used in combination, with the most effective combination being the Jak inhibitor (CP-690550) and Flt3 inhibitor (tandutinib) in M-phase readout.

• AraC + daunorubicin together or bortezomib alone demonstrated complete M-phase inhibition and induction of apoptosis compared with vehicle control (>60% versus 25%, respectively). However, apoptosis was induced in the non-tumor lymphoid cells as well. Conclusions: This case study provides initial proof-of-concept that SCNP can be used to dissect the pathophysiologic heterogeneity of hematologic tumors and assess their differential response to single and combination therapies. Results were rapid with adequate turnaround time for clinical applications. Ultimately, this functional pathway profiling and drug sensitivity assay could be used to guide treatment decisions and improve patient management.

This talk is also presented as Poster B16.