Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells.

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 µm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.