Human acute myeloid leukemia (AML) cell line LFQ, comparison between doxycycline-inducible control shRNA and Pim2 shRNA

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies including FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. MOLM-14 is a human FLT3-ITD AML cell line that was transduced with control (scrambled) or anti-Pim2 shRNA using lentivirus. Expression of hairpins was induced by doxycycline during 3 days and the experiments were done in biological triplicates. One million of MOLM-14 shSCR or shPim2 cells were lysed in 200µl lysis buffer (50 mM Tris (pH 8.5), 2% SDS, 20 mM TCEP, 50 mM chloroacetamide) and heated 5 min at 95°C. Proteins (50µg) from whole cell lysate were trypsin-digested overnight using the filtered-aided sample preparation (FASP) method. Liquid chromatography and mass spectrometry analyses were performed on anU3000 RSLC nanoflow-HPLC system coupled to a Q-Exactive Orbitrap mass spectrometer (both from Thermo Fisher Scientific). The analyses of raw data were performed by LFQ on Maxquant software with a maximum of 1% FDR and match between runs enabled within a 0.7 min match time window and 20 min alignment time window. Maxquant LFQ data were analyzed with Perseus, Ingenuity and Excel softwares.