PO-114 Histone deacetylase 6 regulates NOTCH3 endosomal trafficking and degradation in T-cell acute lymphoblastic leukaemia cells

T-ALL is a malignancy characterised by aberrant Notch signalling, sustained by activating mutations in Notch1 as well as over-expression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Given some limitations of existing drugs blocking Notch signalling, it is important to get new insights into the biology of Notch3 to further stimulate the development of Notch-targeted therapies in cancer. T-ALL cell lines and cells obtained from patient-derived xenografts (PDX) were treated in vitro with HDAC inhibitors and other drugs or HDAC6-specific shRNA and effects on Notch expression and activity were investigated by standard methods. Confocal microscopy was used for intracellular localization studies. PDX models were also utilised to investigate effects of HDAC6 silencing on leukaemia growth. We initially found that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTa, CR2 and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of Dynein with Ciliobrevin D. Pharmacologic studies with HDAC1, 6 and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukaemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels by a mechanism involving endosomal sorting and suggest HDAC6 as potential novel therapeutic target to lower Notch signalling in T-ALL and other Notch3-addicted tumours.