T-cell Checkpoint Pathways Modulate Cell Cycle and Steroid Resistance in T-cell Acute Lymphoblastic Leukaemia

Abstract

T-cell Acute Lymphoblastic Leukaemia (T-ALL) results from the malignant transformation of immature T cells with hallmarks of differentiation blockage and expansive proliferation. The checkpoints during T-cell development are provided by the pre-T-Cell Receptor (pTCR) for β-selection and T-Cell Receptor (TCR) for positive and negative selection. Recurrent gene lesions in proximity to pTCR/TCR are found in T-ALL, such as NOTCH1, FBXW7 and PTEN. Kinases of the pre-B-cell receptor signalling have been validated as drug targets in B-ALL. Whether any critical components of T-cell checkpoint pathways, such as Lymphocyte-specific non-receptor tyrosine Kinase (LCK), play a comparable role in T-ALL remains unclear. A targeted shRNA screen against central components of the pTCR/TCR signalling complex in TALL cell lines in vitro and primary cells in vivo identified an essential role for LCK in cell proliferation and leukaemia propagation. Knockdown of LCK in the cell lines SUPT1, CUTLL1 and MOLT4 revealed a substantial growth defect over time. This was further validated in a competitive assay using MOLT4 in vivo, in which control cells outcompeted LCK knockdown cells in mouse bone marrow, spleen and liver. Phenotypically, knockdown or inhibition of LCK by dasatinib (DAS) impaired cell proliferation by inducing G0/G1 arrest in T-ALL cell lines and patient-derived xenografts (PDXs) with only trivial induction of apoptosis. The sensitivity of T-ALL cell lines to DAS significantly correlated with LCK activation (pY394LCK/LCK). Several cell lines and PDXs were glucocorticoid-resistant in vitro. Interestingly, T-ALL cells were resensitised to dexamethasone (DEX) after LCK knockdown. Correspondingly, DAS reinforced DEX-induced apoptosis. Drug matrix analyses of ten T-ALL cell lines and six PDXs revealed a significant enrichment of DEX/DAS synergy at clinically relevant concentrations. A murine phase ‖-like clinical trial of ten T-ALL PDXs confirmed that DEX/DAS significantly impaired leukaemia engraftment. Engraftment of human leukaemia cells in spleen, bone marrow, and liver was substantially decreased in the DEX/DAS group compared with control or single-drug therapy. In addition, DEX/DAS significantly reduced CNS infiltration of leukaemia cells. The presented research outcomes highlight the crucial role of LCK in T-ALL proliferation and propagation. LCK inhibitor DAS, in conjunction with DEX, reverses steroid resistance and significantly reduces leukaemia propagation in vivo. The DEX/DAS combination might provide a novel therapeutic strategy for refractory and relapsed T-ALL patients.