Abstract LB-325: Global analysis of the phosphoproteome of human blasts reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib
Authors: Schaab Christoph , Oppermann Felix , Klammer Martin , Pfeifer Heike , Tebbe Andreas , Oellerich Thomas , Krauter Juergen , Levis Mark , Perl Alexander , Daub Henrik , Steffen Bjoern , Godl Klaus , Serve Hubert 
Published: May 2015

Abstract
Acute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. Quizartinib (AC220) is an example of a potent FLT3 inhibitor that was studied in a recent phase II open-label study in patients with relapsed/refractory AML. However, the presence of activating mutations within FLT3 can predict response to a certain extent only.
Here, we investigated whether large-scale analyses of phosphorylation-based signalling events allows identification of more accurate markers based on the hypothesis that the read-out is closer to the mode of action of FLT3 inhibitors. Therefore, we applied quantitative mass-spectrometry to globally profile the phosphoproteome of 12 pre-treatment bone marrow aspirates obtained from AML patients treated with the quizartinib. A signature derived from this analysis consists of five phospho-sites within the proteins EEPD1, BCL11A, RANBP3, RP3, and LMN1 and it accurately predicted response to treatment with AC220 as revealed by validation in additional independent nine AML patients.
Although the combined signature of five phospho-sites showed the highest prediction accuracy, we could demonstrate that in particular phosphorylation of S640 on BCL11A and S333 on RANBP3 lead to almost equally good predictions if used as individual markers. Furthermore, we could show that in case of BCL11A, EEPD1, and LMN1 the expression of the total protein correlates with its phosphorylation and thus with response. The phosphorylation markers were identified and validated in bone marrow aspirates. Although, it is clinical standard procedure to use bone marrow aspirates for diagnosis of AML patients, a predictive test that can be applied to peripheral blood samples would have many advantages. Indeed, we could show that the phosphorylation of the marker proteins strongly correlate between bone marrow and peripheral blood samples from the same patients, suggesting that the phosphorylation or protein markers can be measured and are predictive in both, bone marrow and peripheral blood samples.
Citation Format: Christoph Schaab, Felix Oppermann, Martin Klammer, Heike Pfeifer, Andreas Tebbe, Thomas Oellerich, Juergen Krauter, Mark Levis, Alexander Perl, Henrik Daub, Bjoern Steffen, Klaus Godl, Hubert Serve. Global analysis of the phosphoproteome of human blasts reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2014-LB-325