Proteome and phosphoproteome changes associated with prognosis in acute myeloid leukemia

Abstract

word count: 213 Number of figures and tables: 5 Number of references: 84 Key Points  V-ATPases and RNA processing proteins are downregulated and upregulated, respectively, in relapse patients  Relapse patients show higher activity of CSK2 and CDKs when compared to relapse-free patients AbstractAcute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly.Although complete remission (CR) is achieved for majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has therefore become of major clinical interest in AML.We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a 5-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells.This suggests that therapy against the upregulated kinases also could target the factors inducing their upregulation rather than their activity. In conclusion, our study presents markers that could help predict AML relapse and direct therapeutic strategies.

The Progression of Acute Myeloid Leukemia from First Diagnosis to Chemoresistant Relapse: A Comparison of Proteomic and Phosphoproteomic Profiles

“…To ensure that our patients were comparable, all samples included in the present study had to fulfill the following criteria: (i) a high percentage of AML cells among peripheral blood leukocytes both at the time of first diagnosis and at the time of first relapse; (ii) enriched AML cell populations including at least 90% of leukemic cells (documented both by microscopy and by flow cytometry) could thereby be prepared by highly standardized density gradient separation of viable cell suspensions [ 20 ]; (iii) all samples were thus derived from the same in vivo compartment, i.e., peripheral blood; and (iv) ex vivo handling of all blood samples was in accordance with the same standardized guidelines. We could thereby ensure a similar and high quality of all first diagnosis and first relapse samples included in the present study.…”

“…The present DIAGNOSIS–FIRST RELAPSE proteome and phosphoproteome datasets, based on the super-SILAC mix results, were compared with those described in a larger cohort of AML samples collected at the time of first diagnosis from patients that either relapsed or had been AML-free for at least 5 years [ 20 ]. Few proteins and phosphorylation sites overlapped in both studies ( Figure S2a,b ), showing that the proteome and phosphoproteome changed considerably from the first diagnosis to the first relapse.…”

“…We have recently reported the proteomic and phosphoproteomic profiles of AML cells derived from patients at the first diagnosis before any antileukemic treatment [ 20 ]. The paired DIAGNOSIS–FIRST RELAPSE samples of the present study revealed significant alterations of the proteome and phosphoproteome between primary (diagnosis point) AML cells and the relapsed AML cells.…”

“…RNA transcription and translation processes were also upregulated in the FIRST RELAPSE group. This observation is especially significant, since the levels of proteins associated with RNA transcription and translation were higher already at diagnosis for those patients that did relapse later [ 20 ]. It appears, therefore, that a high AML cell capacity for protein translation is associated with an increased relapse risk.…”

“…Various molecules involved in survival, apoptosis, and metabolism were also modulated, but these observations were patient-specific. In a previous study, we utilized LC–MS/MS-based proteomics/phosphoproteomics and the super-SILAC (Stable Isotope Labeling with Amino acids in Cell culture) mix quantitation approach to compare the proteome and phosphoproteome of AML cells derived at the time of first diagnosis from patients who later became leukemia-free survivors to those of AML cells acquired from patients who relapsed after an initial intensive and potentially curative treatment [ 20 ]. In our present study, we used the same methodological approach to compare proteomic and phosphoproteomic profiles for paired samples derived at the first time of diagnosis and at later first relapse.…”