RUNX2 as a dependency factor and critical mediator of disease progression in high-risk T-ALL

Introduction

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive variant of ALL with very rapid progression and poor prognosis that primarily affects children, adolescents, and young adults. The high-dose chemotherapies used to treat T-ALL are often highly toxic and life-threatening.¹

T-ALL can be classified by unique gene expression signatures corresponding to different stages of T-cell developmental arrest, characterized by aberrant regulation of specific transcription factor oncogenes including, among others, the HOXA oncogene. HOXA overexpression is a feature of KMT2A-rearranged (KMT2A-R) T-cell leukemia and is considered a factor in the poor prognosis and reduced survival of immature leukemias.¹

Runt-related transcription factor 2 (RUNX2) is a pivotal regulator for osteoblast and chondrocyte differentiation and bone development. There is limited evidence on the role of RUNX2 in normal hematopoiesis and T-cell development. A recently published study by Matthijssens et al.¹ investigated the complex relationship of RUNX2 in the pathobiology of T-ALL. The key findings are summarized below.

Study design

The study used multiple analyses on several in vivo and in vitro datasets to explore the role of RUNX2 as a dependency factor and mediator of disease progression in high-risk T-ALL.

Results

RUNX2 is highly expressed in immature and KMT2A-R T-ALL

• RUNX2 presented significantly (p < 0.05) higher expression levels in HOXA and LYL1 subtypes compared with other T-ALL subtypes.
• Patients with early T-cell precursor (ETP) showed significantly higher levels of RUNX2 mRNA compared with patients with near-ETP and non-ETP.

RUNX2 is upregulated in T-ALL harboring KMT2A-R and/or immature/ETP phenotype

• KARPAS-45, PER-117, and LOUCY showed the highest levels of RUNX2 expression.
• T cells with an immature phenotype showed the highest levels of RUNX2 mRNA along with one sample with a cortical immunophenotype and a KMT2A-MLLT1 translocation.
• Cell samples from patients with KMT2A-R T-ALL indicated higher levels of RUNX2 mRNA and protein compared with samples without such lesions.

Runx2 ablation does not affect normal hematopoiesis or T cell development

• RUNX2 mRNA levels were highest in CD34+ T cells and sharply decreased toward the CD4⁺CD8⁺ double-positive stage. Similarly, these were highest in ETP cells and gradually decreased over the different maturation stages.
• There were no significant differences in white blood cell (WBC), lymphocyte, T-cell subsets, neutrophil, red blood cell (RBC), hemoglobin or platelet counts in samples of heterozygous, and homozygous Runx2 knockout mice.

RUNX2 essential for the survival of immature and KMT2A-R T-ALL

• RUNX2 depletion lead to an increase in the number of early apoptotic cells and delayed cell cycle progression in S and G₂/M phases, while there was an increase in G₁/G₀.
• RUNX2 depletion led to a significant decrease in the levels of active β-catenin and survivin, known downstream targets of AKT signaling.

RUNX2 regulates T-ALL engraftment and disease progression

• Mice inoculated with RUNX2-depleted cells showed prolonged survival and decreased levels of leukemic cells infiltrating the bone marrow (BM), spleen, and meninges.

• KMT2A-MLLT1 PDX cells transduced with RUNX2 shRNA showed GFP-positive cells were absent or significantly reduced in the peripheral blood, BM, and spleen.

RUNX2 directly regulates an oncogenic transcription signature

• The most significantly enriched motif was the RUNX-binding motif in both cell lines suggesting that RUNX2 acts in harmony with other hematopoietic transcriptional regulators.
• High affinity was observed for binding of KMT2A fusion proteins on the RUNX2 promoter in both human and mice cells.

RUNX2 promotes T-ALL cell migration and adhesion in vitro

• Overexpression of RUNX2 increased the expression of the CXCR4 receptor and enhanced T-ALL cell migration to the CXCL12 chemokine.
• RUNX2 enhanced the expression of proteins linked to epithelial-mesenchymal transition (EMT), and the adhesion of T-ALL cells to fibronectin and increased the expression of VLA-4.

RUNX2 exacerbates T-ALL progression and extramedullary infiltration

• The disease progression was accelerated in mice engrafted with PF382 and RUNX2 overexpressing (RUNX2 OE). The median survival was decreased in RUNX2 OE group by 38% (p < 0.0001).
• Elevated levels of several chemokines, including CXCL10, CCL2, CCL4, CCL5, IL-6 cytokine, were observed while the levels of IL-1α decreased, suggesting a role of RUNX2 in regulating cytokine signaling and host immune response.

RUNX2 increases metabolic activity of T-ALL cells

• Increased levels of extracellular acidification rate (ECAR), glycolytic flux, glucose uptake, and expression of the glucose transporter GLUT1 were observed with forced expression of RUNX2, suggesting its role in regulating glycolytic metabolism in KMT2A-R and immature leukemias.

RUNX2 regulates mitochondrial dynamics and biogenesis

• RUNX2 upregulation increased mitochondrial membrane potential and reduced reactive oxygen species (ROS) levels in the cell lines.
• When mitochondrial fission or AMPK activation was inhibited, it reduced the ability of T-ALL cells to migrate to CXL12, suggesting the role of RUNX2 in facilitating T-ALL chemotaxis.

Immature T-ALL and KMT2A-rearranged leukemias are vulnerable to pharmacological targeting

• KMT2A target genes such as Hoxa10, Hoxa11, and Hoxb13, showed reduced expression in RUNX2-deficient KMT2A-R cells.
• Cell lines KARPAS-45 and PER-117 were both sensitive to AI-10-104 treatment, suggesting that AI-10-104 acts as a RUNX2 inhibitor in these cells.
• RNA-Seq on KARPAS-45 cells showed gene sets related to mitochondrial membrane and metabolism were highly enriched in genes that were significantly downregulated by AI-10-104 treatment.

Conclusion

The study identified RUNX2 as a transcription factor that directly and indirectly activates and suppresses a broad spectrum of genes in high-risk T-ALL with KMT2A-R or immature/ETP immunophenotype. RUNX2 was identified as a direct target of the KMT2A chimeras. RUNX2 is essential for the survival of immature and KMT2A-R T-ALL cells in vitro and in vivo and isresponsible for cell cycle, metabolism, mitochondrial dynamics, biogenesis, and T-cell signaling, explaining its prosurvival role in T-ALL. The study also indicated the role of RUNX2 and its downstream targets for therapeutic intervention in immature and KMT2A-R T-ALL.