PO-292 Leukemic T cells stimulate NF-κB signalling in stromal cells through lymphotoxin-β receptor stimulation

ABSTRACT Introduction T-cell acute lymphoblastic leukaemia (T-ALL) is a malignancy arising from T-cell progenitors. The molecular crosstalk between thymocytes and stromal cells is key for T-cell development. Although T-ALL cells require stromal cell support to be maintained ex vivo, it is unclear how thymic microenvironmental cues support T-ALL. We previously showed that stromal lymphotoxin-β receptor (LTbR) favours thymic T-ALL development in Em-TEL-JAK2 transgenic mice. Mouse leukemic cells expressed lymphotoxin (LT) proteins and T-ALL development was impaired in Em-TEL-JAK2 mice lacking the Ltbr gene or treated with an LT antagonist. These results suggest that the LT-LTbR signalling axis mediates the crosstalk between malignant and non-malignant cells, thus favouring leukemogenesis. Material and methods To test whether LT-expressing leukemic cells can activate LTbR in stromal cells we use an in vitro coculture system. Since the main signalling pathway activated by LTbR is that leading to NF-κB transcription factor activation, we have generated luciferase reporter cell lines, by transducing LTbR-expressing stromal cell lines (NIH3T3 fibroblasts and MS5 bone marrow stromal cells) with a lentivirus carrying the luciferase reporter gene linked to an NF-κB promoter. The validation of the in vitro reporter cellular system was performed by LPS and agonist anti-LTβR treatment. For coculture assays, primary leukemic cells from Em-TEL-JAK2 mice were seeded on top of reporter stromal cells. For LT-blocking experiments, a soluble LTbR-Fc fusion protein was used. Results and discussions Our results demonstrate that LPS and anti-LTβR activate the NF-κB-luciferase reporter in both cell lines. More importantly, mouse leukemic cells activated the NF-κB reporter in the MS5 and NIH3T3 cells. Showing that NF-κB activation was mediated through LTbR stimulation, luciferase activity in co-cultures was blocked by soluble LTbRFc protein. In addition, NF-κB reporter induction by co-cultured leukemic cells was impaired in LTbR-deficient mouse embryonic fibroblasts. We are currently generating LTbR knockout (KO) in MS5 and NIH3T3 stromal cell lines by CRISPR/Cas9 which will also carry the NF-κB-luciferase reporter. The LTbR-KO and WT stromal cells co-cultured with leukemic cells will be sorted for RNA-Seq analysis to identify the specific LTbR-dependent transcriptional program. Conclusion In conclusion, LT-expressing leukemic cells can activate LTbR signalling in neighbouring stroma cells.