Mitochondrial hyperactivity predicts chemotherapy relapse and venetoclax resistance in pediatric B-cell acute lymphoblastic leukemia Free

Relapse remains the major cause of treatment failure and mortality in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Venetoclax, a selective BCL-2 inhibitor approved for adult myeloid malignancies, has recently demonstrated promising activity in relapsed/refractory pediatric leukemia. However, the molecular basis of venetoclax resistance in ALL is incompletely understood, particularly in the context of pediatric disease. Mounting evidence highlights the role of mitochondrial metabolism in therapeutic resistance across hematologic malignancies, but its role in childhood B-ALL has yet to be fully delineated.

We conducted a prospective multicenter Phase Ib/II clinical trial (ChiCTR2200062592) evaluating the safety and efficacy of venetoclax in combination with chemotherapy in relapsed pediatric ALL. Twenty-seven B-ALL patients were enrolled in the dose exploration stage from 2021 to 2022. Venetoclax was administered at 240 mg/m²/day (adult equivalent 400 mg/day) for two consecutive blocks. Comprehensive molecular profiling was performed on paired longitudinal samples (diagnosis, chemotherapy relapse/pre-venetoclax [Rel/preVEN], and venetoclax-resistant/post-treatment relapse [VEN-Rel]) from 16 patients. We applied single-cell RNA-seq and BCR-seq, along with bulk whole-exome and transcriptome sequencing. Functional genomic screening using genome-wide CRISPR-Cas9 knockout libraries was conducted to identify resistance drivers. Enrichment analysis of CRISPR-depleted hits was performed to investigate pathway-level vulnerabilities.

Among the 27 patients enrolled, 26 (96.3%) responded to venetoclax-based therapy, with 23 (85.2%) achieving complete remission after the first cycle. Fifteen patients attained MRD negativity. At a median follow-up of 22 months, 22 patients remained in durable remission after receiving consolidation with immunotherapy or transplantation.

Single-cell multi-omics analysis revealed marked upregulation of mitochondrial oxidative phosphorylation (OXPHOS), electron transport chain (ETC) activity, and ATP biosynthesis in relapse and Rel samples, compared to diagnostic samples. Apoptosis-related pathways were simultaneously downregulated. These features were validated in a large diagnostic cohort of 426 pediatric B-ALL patients (CCCG-ALL-2015) using NetBID2-based activity inference. We constructed two transcriptional activity scores: a “MitoScore” reflecting mitochondrial respiratory activation, and an “ApopScore” representing intrinsic apoptotic signaling. Across molecular subtypes, high MitoScore was associated with early MRD persistence (Day 19), increased cumulative incidence of relapse (CIR), and inferior event-free and overall survival. In contrast, high ApopScore correlated with favorable outcomes. These observations were further validated in two independent external cohorts (MaSpore/LLAG and TARGET-ALL).

Comparison of venetoclax responders (n=8) and poor responders (n=7) revealed that poor responders harbored transcriptionally more differentiated leukemic populations (pro-B-like and pre-B-like), enriched for mitochondrial activity. GSEA of bulk and single-cell transcriptomes showed consistent enrichment of OXPHOS and nucleoside triphosphate metabolic pathways in poor responders. Post-venetoclax samples further demonstrated clonal selection of OXPHOS-high subpopulations, suggesting mitochondrial hyperactivity as a hallmark of therapeutic resistance.

Finally, unbiased genome-wide CRISPR screening identified genes involved in mitochondrial function as significantly depleted in venetoclax-treated leukemia models, implicating mitochondrial energetics as central to drug response.

Our integrative clinical and molecular study—based on the venetoclax-treated pediatric B-ALL cohort—demonstrates that mitochondrial hyperactivity is a convergent mechanism driving both chemotherapy and venetoclax resistance. Mitochondrial metabolic states correlate with differentiation trajectories, clonal fitness, and treatment outcomes. These findings provide a mechanistic framework for precision risk stratification and lay the foundation for rational design of mitochondrial-targeted combination therapies in relapsed pediatric B-ALL.