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

Abstract

Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Nearly 50% of the patients who receive the most intensive treatment develop chemoresistant leukemia relapse. Although the leukemogenic events leading to relapse seem to differ between patients (i.e., regrowth from a clone detected at first diagnosis, progression from the original leukemic or preleukemic stem cells), a common characteristic of relapsed AML is increased chemoresistance. The aim of the present study was to investigate at the proteomic level whether leukemic cells from relapsed patients present overlapping molecular mechanisms that contribute to this chemoresistance. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the proteomic and phosphoproteomic profiles of AML cells derived from seven patients at the time of first diagnosis and at first relapse. At the time of first relapse, AML cells were characterized by increased levels of proteins important for various mitochondrial functions, such as mitochondrial ribosomal subunit proteins (MRPL21, MRPS37) and proteins for RNA processing (DHX37, RNA helicase; RPP40, ribonuclease P component), DNA repair (ERCC3, DNA repair factor IIH helicase; GTF2F1, general transcription factor), and cyclin-dependent kinase (CDK) activity. The levels of several cytoskeletal proteins (MYH14/MYL6/MYL12A, myosin chains; VCL, vinculin) as well as of proteins involved in vesicular trafficking/secretion and cell adhesion (ITGAX, integrin alpha-X; CD36, platelet glycoprotein 4; SLC2A3, solute carrier family 2) were decreased in relapsed cells. Our study introduces new targetable proteins that might direct therapeutic strategies to decrease chemoresistance in relapsed AML.

Keywords: CDK; acute myeloid leukemia; degranulation; kinase; markers; mass spectrometry; minimal residual disease; mitochondria; patient relapse; phosphoproteome; proteome; secretion.

Figure 1

Overview of the matched diagnosis–first relapse patient cohort. The study included paired acute myeloid leukemia (AML) cell samples from seven patients, collected at the times of first diagnosis (DIAGNOSIS) and first relapse (FIRST RELAPSE). All patients received intensive induction chemotherapy and consolidation therapy and achieved complete remission (CR) (Table 1). All relapses occurred within three years after CR.

Figure 2

The AML proteome and phosphoproteome are enriched in mitochondrial ribosomal, RNA-processing, and DNA repair proteins at first relapse. Gene ontology (GO) analysis was conducted for the proteome (a) and phosphoproteome (b), separately. Top 10 enriched GO categories are shown on the y-axis of each bar plot. The -log₁₀ p values and the number of proteins associated with these GO categories are shown on the X-axis. Dashed bars represent GO categories that are also significantly enriched with false discovery rate (FDR) < 0.05. (c,d) Networks of protein–protein interactions (PPI) based on STRING search of the proteomic and phosphoproteomic datasets and visualized in Cytoscape after ClusterONE analysis. Significance of high cohesiveness of protein and phosphoprotein networks is shown by the p value of a one-sided Mann–Whitney U test. Fold changes (FCs) of protein expression or phosphorylation according to the super-SILAC (Stable Isotope Labeling with Amino acids in Cell culture) mix method are color-coded; orange-colored proteins showed a higher expression or phosphorylation in the FIRST RELAPSE group, and Yale blue-colored proteins showed a higher expression or phosphorylation in the DIAGNOSIS group. The differentially regulated phosphorylation site(s) is shown next to each phosphoprotein.

Figure 3

AML phosphoproteome in the FIRST RELAPSE group is enriched with cyclin-dependent kinases (CDKs) substrates. (a) Sequence motif analysis of the ± 31 amino acids flanking the differentially regulated phosphorylation sites (indicated with a black arrow) for the FIRST RELAPSE and DIAGNOSIS groups. (b) Kinase-substrate enrichment analysis (KSEA) of differentially regulated and unregulated phosphorylation sites. The kinase z-score (X axis) is the normalized score for each kinase (Y axis), weighted by the number of identified substrates.