P350: dab1 expression drives proliferation and metabolic activity in t-all and interferes with t-cell-specific pathways

Background: Our integrative analyses of RNA-Seq and ATAC-Seq datasets previously identified DAB1 to be upregulated and highly accessible in the majority of pediatric T-ALL patients (79%) when compared to normal T-cells. The role of DAB1 is unexpected because it encodes an adaptor protein of the Reelin signaling pathway, which regulates neuron migration and differentiation in the brain. Previous studies have indicated a non-canonical interaction between DAB1 and Notch signaling pathway, which is constitutively active in >60% of pediatric T-ALL patients. Aims: We aimed at a functional analysis of DAB1 on the survival, proliferation, and gene expression profile of T-ALL cells. Methods: We performed RNA-Seq analysis of six T-ALL cell lines and identified genes highly correlating with DAB1 expression by over-representation analysis (ORA). Two cell lines (Jurkat: DAB1-low and RPMI-8402: DAB1-high) were selected for further functional experiments. DAB1 was inactivated by CRISPR/Cas9 to generate knock-out (KO)-clones. BrdU-, Annexin-V- and MTT assays were performed in DAB1-inactivated and in unmodified control cells. Changes in gene expression profiles of KO-clones were assessed by RNA-Seq. Results: Over-representation analysis revealed that DAB1 RNA-expression in the six T-ALL cell lines correlated (r≥0.9) with the biological processes lymphocyte activation and regulation of response to cytokine exposure (FDR<0.05; WebGestalt). These correlating gene sets revealed significant enrichment of genes that are highly relevant in the context of T-cell leukemogenesis such as PTEN, RUNX3, IL6R and kinases (JAK3, MAP3K8, TXK). BrdU proliferation assays showed that KO-clones of the DAB1-high expressing line RPMI-8402 exhibit a significant decrease of cells undergoing S-phase (p≤0.02) when compared to wildtype cells (WT). In addition, all three KO-clones of this line showed a significant reduction in metabolic activity (MTT assay, p≤0.03) and significantly higher number of apoptotic cells in comparison to WT-cells (Annexin V-assay, p≤0.03). When we compared KO-clones of the DAB1-low cell line Jurkat to the corresponding WT, we did not identify any significant differences in cell proliferation, metabolic activity and apoptosis. To understand the effects of DAB1 loss in DAB1-high T-ALL cells, we ran differential expression analysis and found 32 genes up- and 45 genes down-regulated in RPMI-8402 KO-clones when compared to WT (padj<0.05; DESeq2). These genes were significantly enriched for those involved in T-cell activation, regulation of T-cell proliferation, and leukocyte differentiation (FDR<0.05; WebGestalt). The upregulated genes included IL2, IL10, ITK (IL2-inducible kinase), TNFRSF14 and RASAL3 (Ras activator like 3). Another potential effector upregulated in KO cells was tumor suppressor DHRS2 – a target of LEF1 which is inactivated by mutations in a subset of T-ALL patients. Further, we also identified significant downregulation of NOTCH3 and PTCRA in KO-clones, both of which play pivotal roles in T-cell development and T-ALL leukemogenesis. PRDM16 - a transcription regulator essential for HSC maintenance and found to support leukemogenesis in AML - correlated with DAB1 expression in the six T-ALL cell lines and was downregulated upon DAB1 inactivation. Summary/Conclusion: Our results demonstrate that DAB1 expression plays a functional role in a subset of T-ALLs by driving proliferation and metabolic activity in T-ALL cells, which may indicate a previously unknown susceptibility of this type of leukemia. Keywords: T-ALL