Metabolic Signature of Oxidative Phosphorylation Deficiency with Activated Nucleotide Biosynthesis in Early T-Cell Precursor Acute Lymphoblastic Leukemia

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) represents a particularly aggressive subtype of T-ALL characterized by stem cell-associated properties. Despite its clinical significance, comprehensive studies elucidating the proteomic background underlying this distinct subgroup have been lacking. In this study, we present an in-depth 4D proteome quantification analysis of bone marrow specimens obtained from eight patients newly diagnosed with ETP-ALL. For comparative purposes, samples from patients with T-ALL, AML, and healthy donors (HD), matched for age and sex (n=8 per group), were included to delineate the unique proteomic signature of ETP-ALL.

A total of 8,119 distinct protein groups were identified, with 1,063 differentially expressed proteins between T-ALL and AML, whereas fewer differential proteins (n=686) were found between ETP-ALL and AML, reflecting the immature leukemic status of ETP-ALL, which resembles myeloid progenitor cells. Notably, ETP-ALL exhibited distinctive regulation of ATP synthesis coupled with electron transport and oxidative phosphorylation (OxPhos) compared to AML and T-ALL. Through k-means clustering, we identified six expression clusters, with cluster 4 being the most enriched, particularly associated with OxPhos.

Our analysis identified 24 featured OxPhos proteins that distinguish ETP-ALL from non-ETP-ALL in various datasets (TARGET; Zhang et al., Nature, 2012; Dai et al., PNAS, 2012). We further validated the expression of three representative ETC complex proteins—NDUFB6, NDUFS5, and COX7C—which were significantly attenuated in ETP-ALL compared to T-ALL and AML. Additionally, ETP-ALL exhibited weakened activity of cytochrome C oxidase and decreased cellular total NAD(H). More importantly, primary ETP-ALL cells demonstrated a reduced oxygen consumption rate and mitoATP production. Collectively, ETP-ALL cells displayed metabolic pathways akin to immature progenitors or stem cells, characterized by low OxPhos activity and a quiescent status. We discovered that ETP-ALL uniquely displays a deficiency in ETC-mediated OxPhos, suggesting this as a potential diagnostic marker alongside immunophenotypic criteria. This observation also suggests a promising therapeutic strategy that increases OxPhos activity to alter cell metabolism while concurrently inducing differentiation to sensitize cells to chemotherapy.

Moreover, we identified CD109 as a novel immunophenotypic marker capable of distinguishing ETP-ALL from other T-ALL subtypes and AML. Validation in a cohort of primary samples showed that the positive percentage of CD109 was around 15-20% in ETP-ALL patients, which is dim expressed (positive ratio <5%) in T-ALL or AML, implying its potential diagnostic utility. CD109 is known as a probably one of stem cell markers, which may also relate to low OxPhos activity and a quiescent status characteristics of ETP-ALL.

To further elucidate the distinct metabolic pathways in ETP-ALL, we identified significantly differentially expressed proteins involved in metabolic pathways across ETP-ALL, T-ALL, AML, and healthy MNCs. Each group exhibited distinct metabolic signatures, highlighted by 123 feature proteins. We projected these features across the 32 samples into a 2-dimensional landscape using UMAP and observed that the metabolic features were sufficient to cluster patients and healthy donors according to their subtypes. Notably, ETP-ALL was positioned between AML and T-ALL, consistent with previous reports. Interestingly, all key enzymes involved in nucleotide biosynthesis, particularly de novo purine biosynthesis, exhibited heightened expression in ETP-ALL cases. Our findings not only reveal the metabolic signature of ETP-ALL but also underscore the potential of targeting purine metabolism as a novel therapeutic avenue for ETP-ALL. The distinctive activation of the nucleotide biosynthetic process in ETP-ALL suggests a promising therapeutic target.

ETP-ALL identification currently relies solely on immunophenotypic criteria, potentially overlooking cases due to genetic heterogeneity. Our study unveils a distinct global protein profile, metabolic signature, and a novel marker specific to ETP-ALL, providing new insights into diagnostic criteria and offering potential avenues for clinical management and therapeutic intervention.