Early changes in rpS6 phosphorylation and BH3 profiling predict response to chemotherapy in AML cells.

Abstract

Blasts from different patients with acute myeloid leukemia (AML) vary in the agent(s) to which they are most responsive. With a myriad of novel agents to evaluate, there is a lack of predictive biomarkers to precisely assign targeted therapies to individual patients. Primary AML cells often survive poorly in vitro, thus confounding conventional cytotoxicity assays. The purpose of this work was to assess the potential of two same-day functional predictive assays in AML cell lines to predict long-term response to chemotherapy. (i) Ribosomal protein S6 (rpS6) is a downstream substrate of PI3K/akt/mTOR/ kinase and MAPK kinase pathways and its dephosphorylation is also triggered by DNA double strand breaks. Phospho-rpS6 is reliably measurable by flow cytometry and thus has the potential to function as a biomarker of responsiveness to several therapeutic agents. (ii) A cell's propensity for apoptosis can be interrogated via a functional assay termed "Dynamic BH3 Profiling" in which mitochondrial outer membrane permeabilization in drug-treated cells can be driven by pro-apoptotic BH3 domain peptides such as PUMA-BH3. The extent to which a particular cell is primed for apoptosis by the drug can be determined by measuring the amount of cytochrome C released on addition of BH3 peptide. We demonstrate that phospho-rpS6 expression and PUMA-BH3 peptide-induced cytochrome C release after 4 hours both predict long term chemoresponsiveness to tyrosine kinase inhibitors and DNA double strand break inducers in AML cell lines. We also describe changes in expression levels of the prosurvival BCL-2 family member Mcl-1 and the pro-apoptotic protein BIM after short term drug culture.