Proteomic Characterization of Pediatric Acute Lymphoblastic Leukemias Sheds Light on Subtype-Specific Molecular Mechanisms

Acute lymphoblastic leukemia (ALL) accounts for approximately one third of cancers diagnosed in children every year. Molecular subtyping has significantly improved patient stratification and treatment selection for pediatric leukemia patients, leading to better outcomes. For many ALL subtypes, the molecular mechanisms by which driver mutations promote cancer development and progression remain unknown. We hypothesize that a comprehensive multi-omic profiling strategy, integrating genomic, transcriptomic, and proteomic data, can identify subtype-specific mechanisms and putative treatment options. Here, we present a pilot study comparing the global proteomes of ETV6::RUNX1 and BCR::ABL1-like (Ph-like) pediatric B-ALL patient samples.

For each subtype, we selected 5 patients with frozen blood and/or bone marrow aspirate collected at diagnosis and remission. Cells were lysed, reduced, alkylated, and digested with trypsin overnight following a standard protocol. The peptides were enriched for phosphorylation by the SMOAC method. The peptides that did not bind the resin were analyzed as the global sample and the enriched samples were pooled and run as the phosphopeptide enriched fraction. Samples were injected with a Vanquish Neo (Thermo) onto a PepMap Neo C18 column (75 um x 150 mm, 2um) and eluted over 2 hours with a linear gradient. The column was directly interfaced with the nano electrospray ionization source on the Orbitrap Ascend mass spectrometer (Thermo) equipped with a high field asymmetric ion mobility spectroscopy (FAIMS) source. Intact peptides were collected in the Orbitrap at resolving power of 120, 000 and the MS2 ions were collected in the ion trap using rapid collection (global) or Orbitrap at 30,000 resolving power (enriched). Data were searched in Proteome Discoverer 3.0 against the human protein database downloaded from Uniprot on 05-05-2023. Downstream processing and analysis were performed in R 4.3.3 using proDA 1.16.0 and gProfiler 0.2.3.

We observe a high degree of heterogeneity in the global proteomes of all samples, particularly those collected at diagnosis (83-98% blasts). To identify the most clinically and biologically significant trends, we leveraged the paired nature of our dataset and queried proteins that were differentially expressed (greater than 2-fold change) between diagnosis and remission for each individual patient. Subsequently, we performed pathway enrichment analysis for each patient. Comparing these findings across patients revealed both shared and subtype-specific pathway families enriched at diagnosis. Proteins associated with chromatin remodeling processes and cell cycle regulation, including members of the SWI/SNF family, BCL2, and RB1 were more highly expressed in Ph-like diagnosis samples. In contrast, proteins associated with ribosomes and translational regulation, including IGF2BP1, IGF2BP2, and members of the elongation initiation factor 3 complex, were more highly expressed in ETV6::RUNX1 diagnosis samples.

In this study, we use global proteomic analysis of patient samples to identify mechanistic differences between ETV6::RUNX1 and Ph-like pediatric B-ALLs. This comparative analysis suggests that upregulation of distinct regulatory cascades facilitates cancer development and progression in each subtype. Further integration of this dataset with genomic, transcriptomic, and phosphoproteomic data will enable us to develop specific mechanistic hypotheses focused on the involvement and regulation of these proteins in driving pediatric leukemias. This will in turn facilitate the identification of novel biomarkers and treatment targets to improve patient stratification and treatment selection for children diagnosed with leukemia.

Guest:Jazz Pharmaceuticals: Speakers Bureau; Amgen: Current Employment; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Farooqi:Bayer: Honoraria; Pacific Biosciences: Honoraria, Other: travel support; 10X Genomics: Honoraria, Other: travel support.