Proteogenomic characterization of highly enriched viable leukemic blasts in acute myeloid leukemia: A SWOG report

Abstract

Introduction: Acute myeloid leukemia (AML) remains one of the deadliest hematopoietic malignancies. A better understanding of the molecular biology governing AML may lead to improved risk stratification and facilitate the development of novel therapies. Proteins are responsible for much of the biology of cells. Several studies have examined the global proteome in bulk mononuclear cells (MNCs) from AML specimens, which are comprised a heterogenous population of cells at various stages of differentiation.

Methods: Given the potential impact of the nonleukemic cells on protein expression profiles, we applied an integrative proteogenomic approach utilizing next-generation sequencing and mass spectrometry-based proteomics to identify novel protein biomarkers in unsorted MNCs and viable leukemic blasts (VLBs) isolated from blood and bone marrow specimens obtained at the time of AML diagnosis.

Results: We identified significant differences in protein expression between VLBs and MNCs. Subsequent studies (N = 27) focused on proteomic profiling of VLBs that identified novel candidate biomarkers associated with mutational genotypes and clinical outcome, some of which were recapitulated in an independent cohort of patients. Using mass spectrometry, we also detected mutated protein products, some of which were predicted via in silico analyses to be potential neoantigens amenable to adoptive immunotherapy. As previously described, analyses comparing transcript and protein expression showed an overall modest correlation between mRNA and protein dataset, but enriching for genes associated with mutations significantly improved the protein-RNA correlation.

Conclusion: Together, the results provide insight into the biology of VLBs and demonstrate the gains derived from examining the proteome in addition to genome and transcriptome.

Keywords: AML; hematological malignancy; neoantigens; proteogenomics; transcriptomics.

FIGURE 1

Unsupervised hierarchical clustering of identified proteins in three populations of leukemia cells from three different patients. Mononuclear cells (MNCs) containing all leukemic blasts and normal cells from diagnostic specimens from patients 1, 2, and 3. Differentiated leukemic blasts (VLB^CD34− or VLB^CD117−) and undifferentiated leukemic blasts (VLB^CD34+ or VLB^CD117+) from the same patients.

FIGURE 2

Unsupervised hierarchical clustering of proteins identified by tandem mass tag (TMT)–liquid chromatography (LC)–mass spectrometry (MS)/MS in SWOG27 discovery cohort. VLB^CD34+ indicates blasts expressing CD34, VLB^CD117+ indicates blasts that do not express CD34 but have an expression of CD117, VLB^CD45/SSC indicates double negative viable leukemic blasts (VLBs) isolated by low CD45 expression and low side scatter.

FIGURE 3

Detection of mutations at DNA, RNA, and protein levels. (A, B) Figure shows RUNX1 frameshift (FS) mutations in last coding exon identified in two acute myeloid leukemia (AML) patients. DNA (orange boxes) and RNA (blue boxes) levels. (C) FS mutations in last exon (red arrow) change AA sequence eliminating the native stop codon (red arrow), introducing downstream unique AAs and terminating in novel stop codons (purple arrow). Unique peptide sequences from the FS mutations were detected by tandem mass tag (TMT)–liquid chromatography (LC)–mass spectrometry (MS)/MS in two patients: P2008868414 (blue box) and P199814282 (three red boxes).