GSK3ß is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL.

ABSTRACT

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3ß downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3ß silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3ß mutant reduces them. Additionally, the GSK3ß inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3ß in CLL cells. SB216763 reduces GSK3ß/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3ß is less active. We then modulated the activity of two upstream regulators of GSK3ß and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3ß and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3ß, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3ß activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3ß a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.