Mitochondrial ROS Drives HMGB1-Mediated Autophagy and Immune Evasion in MEF2D Rearranged B-ALL

Technical Summary: Mitochondrial ROS Drives HMGB1-Mediated Autophagy and Immune Evasion in MEF2D Rearranged B-ALL

Problem Statement
MEF2D fusion-driven B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a high-risk subtype characterized by poor prognosis, high relapse rates, and resistance to conventional chemotherapy. While previous studies have established that MEF2D fusion proteins disrupt normal B-cell differentiation, the specific molecular mechanisms driving leukemogenesis and immune evasion in this subtype remain elusive. A critical gap exists in understanding how High Mobility Group Box 1 (HMGB1), a chromatin-associated protein essential for V(D)J recombination, is dysregulated in MEF2D-ALL. Specifically, it is unclear whether HMGB1 is merely transcriptionally suppressed or if it undergoes aberrant subcellular redistribution that contributes to leukemic survival and immune escape. Furthermore, the link between mitochondrial dysfunction, reactive oxygen species (ROS), and the transition of HMGB1 from a nuclear cofactor to an extracellular signaling molecule in this context has not been fully elucidated.

Methodology
The study employed a comprehensive multi-omics and translational approach to investigate the pathogenic role of HMGB1 in MEF2D::HNRNPUL1 (MH) fusion-driven B-ALL:

• Multi-Omics Integration: The authors integrated transcriptomic (RNA-seq), proteomic (LC-MS/MS), and chromatin immunoprecipitation sequencing (ChIP-seq) data from MH fusion-positive cell lines to identify HMGB1 as a direct transcriptional target of the fusion protein.
• Cellular and Molecular Mechanisms: Using human leukemia cell lines (REH and Kasumi-9), the study utilized immunofluorescence, Western blotting, and subcellular fractionation to analyze HMGB1 localization. Mechanistic insights were gained through:
  • Mitochondrial Analysis: Transmission electron microscopy (TEM) and Seahorse XF metabolic assays were used to assess mitochondrial morphology and respiratory function.
  • ROS Modulation: Intracellular ROS levels were measured via flow cytometry, and the ROS scavenger N-acetylcysteine (NAC) was used to determine the functional role of oxidative stress.
  • Protein Interactions: The interaction between extracellular HMGB1 and its receptors (RAGE and TLR4) was validated using Proximity Ligation Assay (PLA), Co-immunoprecipitation (Co-IP), Mammalian Two-Hybrid assays, and AlphaFold-based structural modeling.
  • Autophagy Assessment: Autophagy activation was monitored via TEM, LC3B/P62 Western blotting, and pharmacological inhibition (3-MA).
• In Vivo Models:
  • Genetically Engineered Mouse Model (GEMM): A CRISPR/Cas9-generated MH knock-in mouse model was crossed with Cd19-Cre and NRASG12D mice to recapitulate the human disease phenotype, allowing for the analysis of hematopoietic and immune compartment remodeling.
  • Patient-Derived Xenograft (PDX): Primary leukemic cells from MH-positive patients were engrafted into NSG mice to evaluate therapeutic interventions.
• Therapeutic Intervention: The study tested the efficacy of glycyrrhizin (a known HMGB1 inhibitor), either alone or in combination with standard chemotherapy (vincristine and dexamethasone), in the PDX model. Outcomes were assessed via survival analysis, leukemic burden quantification (flow cytometry), and histopathology.
• Immune Profiling: Mass Cytometry (CyTOF) was utilized to characterize the immune landscape and phospho-protein signaling (e.g., pSTAT3) within the bone marrow microenvironment of the GEMM.

Key Results

1. Transcriptional Repression and Nuclear Depletion: Integrative analysis confirmed that the MEF2D fusion protein directly represses HMGB1 transcription. Consequently, MH fusion cells exhibit a significant reduction in total HMGB1 levels, characterized by a specific depletion of nuclear HMGB1 and a concurrent accumulation in the cytoplasm and extracellular space.
2. Mitochondrial ROS as the Driver: MH fusion cells displayed severe mitochondrial dysfunction, including cristae disruption, swelling, and impaired oxidative phosphorylation. This dysfunction led to excessive ROS accumulation. Inhibition of ROS with NAC reversed HMGB1 nuclear export and increased apoptosis, identifying mitochondrial ROS as the upstream driver of HMGB1 redistribution.
3. Autophagy-Immune Evasion Axis: The released extracellular HMGB1 activated RAGE and TLR4 signaling pathways. This activation induced autophagy, which in turn facilitated further HMGB1 secretion, creating a positive feedback loop. This loop enhanced leukemic cell survival and contributed to an immunosuppressive microenvironment.
4. In Vivo Phenotype: The MH/Cd19-Cre/NRASG12D mouse model exhibited hallmark features of B-ALL, including differentiation arrest at the Pre-pro B/Pro B stage, splenomegaly, and extensive remodeling of the immune landscape (e.g., expansion of immature myeloid cells and altered T-cell subsets). Extracellular HMGB1 accumulation correlated with inflammatory infiltration and autophagy activation in vivo.
5. Therapeutic Efficacy: Pharmacological inhibition of HMGB1 using glycyrrhizin significantly reduced leukemic burden, prolonged survival, and decreased organ infiltration in PDX models. While the combination of glycyrrhizin with standard chemotherapy did not significantly extend overall survival compared to chemotherapy alone, it effectively reduced splenic tumor burden and delayed disease relapse following treatment cessation.

Significance and Claims
The paper claims to delineate a novel "mitochondrial ROS-HMGB1-autophagy axis" that drives immune evasion and leukemic progression in MEF2D-rearranged ALL. The authors posit that MEF2D fusion proteins reprogram HMGB1 from a nuclear regulator of B-cell differentiation into an extracellular inflammatory mediator. This dual mechanism—loss of nuclear function (blocking differentiation) and gain of extracellular function (promoting autophagy and immune suppression)—sustains the leukemic state.

The study identifies extracellular HMGB1 as a therapeutically actionable vulnerability. By targeting this pathway with glycyrrhizin, the authors demonstrate a strategy to disrupt leukemic persistence and adaptive resistance within the bone marrow niche, offering a potential avenue to circumvent treatment resistance in this high-risk subtype. The findings suggest that HMGB1 inhibition acts not merely as a cytotoxic agent but as a modulator of the leukemic microenvironment and stress adaptation pathways.