Dysregulation of the inside-out signaling pathway in CNS-infiltrated pediatric T-cell acute lymphoblastic leukemia

Technical Summary: Dysregulation of the Inside-Out Signaling Pathway in CNS-Infiltrated Pediatric T-ALL

Problem Statement
Despite significant improvements in the 5-year overall survival rates for pediatric T-cell acute lymphoblastic leukemia (T-ALL), central nervous system (CNS) infiltration remains a critical barrier to long-term cure. Leukemia cells that migrate to the CNS can evade systemic chemotherapy, leading to relapse and necessitating toxic intrathecal therapies. The molecular mechanisms driving the infiltration of T-ALL blasts into the CNS and their subsequent survival within the CNS microenvironment are not fully understood. Specifically, it is unclear whether the T-cell receptor (TCR)-mediated "inside-out" signaling pathway, which regulates normal T-cell migration via integrin activation (e.g., LFA1 binding to ICAM1), is hijacked by leukemic cells to facilitate CNS invasion.

Methodology
The study employed a multi-faceted approach combining bioinformatic analysis of public datasets with in vitro functional assays:

1. Transcriptomic Analysis: The authors analyzed RNA-sequencing data from 180 pediatric T-ALL bone marrow (BM) samples obtained from the TARGET initiative. Samples were stratified by clinical CNS status: CNS1 (no infiltration), CNS3 (advanced infiltration), and CNS relapse. Differential expression analysis was performed comparing CNS3 and CNS relapse groups against CNS1. Functional enrichment analysis (ORA and GSEA) was conducted using Reactome and Gene Ontology libraries.
2. Comparative Analysis: The T-ALL dataset was compared against healthy hematopoietic stem and progenitor cells (HSPCs) to distinguish disease-specific alterations from general malignancy features.
3. Primary Sample Validation: Primary pediatric T-ALL samples were used to isolate leukemia-initiating cells (LICs, CD34+) and bulk blast cells (CD34-) to validate gene expression patterns via RT-qPCR.
4. Functional Knockdown: Lentiviral shRNA-mediated knockdown of SKAP1 was performed in four T-ALL cell lines (SUP-T1, KARPAS45, CCL-119, MOLT16) to assess the impact on cell proliferation and viability.
5. Co-Culture Models: T-ALL cell lines were co-cultured with human meningeal cells (HMCs) to mimic the CNS niche. These co-cultures were exposed to methotrexate (MTX) to evaluate treatment-induced changes in the inside-out signaling pathway.

Key Contributions and Results

• Transcriptional Signatures of CNS Infiltration:

  • RNA-seq analysis revealed that CNS-infiltrated (CNS3) and CNS-relapsed samples share a distinct transcriptional profile compared to CNS-negative (CNS1) samples.
  • Differentially expressed genes were significantly enriched for pathways related to extracellular matrix (ECM) organization and degradation (e.g., COL9A3, MMP11, TMPRSS6), suggesting a mechanism for tissue invasion.
  • A specific enrichment of the TCR complex and downstream signaling components was observed. Notably, the adaptor protein SKAP1 (also known as SKAP55), a key regulator of inside-out signaling, was consistently upregulated in CNS3 and CNS relapse samples.
  • Other upregulated genes included HINT2 (apoptosis control), HEPACAM, and N-cadherin (CDH2), a marker of epithelial-mesenchymal transition (EMT).

• Specificity of SKAP1 Upregulation:

  • While SKAP1 expression was not significantly different between CNS1 samples and HSPCs, it was significantly upregulated in CNS relapse samples and showed a trend toward higher levels in CNS3 samples compared to HSPCs.
  • In primary T-ALL blasts, SKAP1 and its downstream target ICAM1 were upregulated compared to matched LICs (CD34+).
  • Correlation analysis indicated a positive relationship between SKAP1 and the transcription factor NFATC1 (downstream of TCR) in both CNS3 and CNS relapse groups.

• Functional Impact of SKAP1:

  • Lentiviral knockdown of SKAP1 in T-ALL cell lines resulted in a significant reduction in cell proliferation and viability, particularly in cell lines that grow in clusters (SUP-T1, KARPAS45).
  • SKAP1 knockdown led to the upregulation of ICAM2 and a modest downregulation of the cell cycle regulator Cyclin B1 (CCNB1), suggesting a role for SKAP1 in promoting cell cycle progression and survival.

• Effects of Chemotherapy and Microenvironment:

  • In co-culture with meningeal cells, T-ALL cells showed upregulation of ICAM1 and ALCAM.
  • Upon exposure to methotrexate (MTX) in co-culture, T-ALL cells exhibited a loss of key inside-out signaling components, including LFA1 and ADAP1, and a downregulation of NFATC1.
  • Despite these losses, SKAP1 expression remained unchanged in MTX-treated co-cultures, and a positive correlation between SKAP1 and ICAM2 was maintained. HINT2 was also upregulated in this condition, mirroring findings in clinical samples.

Significance and Claims
The paper concludes that dysregulation of the inside-out signaling pathway, specifically characterized by the upregulation of SKAP1, is a transcriptional feature associated with CNS infiltration and relapse in pediatric T-ALL. The authors claim that:

1. SKAP1 is a potential driver: The upregulation of SKAP1 correlates with increased proliferation and viability in T-ALL cells, suggesting it may be a functional driver of the aggressive phenotype observed in CNS disease.
2. Distinct Molecular Adaptation: The transcriptional changes observed (ECM remodeling, TCR complex enrichment, SKAP1 upregulation) appear specific to CNS-infiltrated disease rather than general T-ALL pathology, as they were not observed in CNS-negative samples when compared to HSPCs.
3. Therapeutic and Diagnostic Potential: The findings suggest that targeting the SKAP1-mediated inside-out signaling pathway could offer new insights for therapeutic interventions or serve as a biomarker for identifying patients at risk of CNS relapse.
4. Chemotherapy Interaction: The study highlights that standard CNS-directed therapy (MTX) alters the signaling landscape of T-ALL cells interacting with the meningeal niche, potentially selecting for cells with specific signaling adaptations.

The authors maintain a modest tone regarding the clinical application, noting that the use of bone marrow samples (rather than direct CSF) is a limitation, and that the functional relevance of these transcriptional programs requires further validation in advanced in vivo models. They emphasize that while the data suggests an association between SKAP1 and CNS infiltration, the precise mechanism of SKAP1 activation in the context of heterogeneous T-ALL subtypes (some lacking surface TCR) warrants further investigation.