Multiple Mechanisms of Notch-Mediated B-ALL Growth Arrest Via HES1 and PLK1: KIF11, CyclinB and p53

Abstract 3137

Notch signaling inhibits B-ALL growth and survival, while it promotes T-ALL, revealing contrasting cell-specific consequences of Notch signaling in ALL subtypes. We previously reported that HES1 (Hairy and Enhancer of split1) is a Notch pathway member which is sufficient to reproduce these effects in B-ALL.

We have found distinct HES1 protein complexes in B-ALL versus T-ALL, with significantly higher molecular weight complexes found in B-ALL. Through characterization of these complexes, multiple novel HES1 interacting proteins have been identified which regulate HES1 function as well as provide growth inhibiting and pro-apoptotic effects. We previously reported the novel role of HES1:PARP1 complexes in inducing B-ALL apoptosis. This study reports Polo-like kinase1 (PLK1) and Kinesin Family member 11 (KIF11, also known as Eg5) as novel HES1 interacting proteins in B-ALL. PLK1 is highly expressed in B cell leukemias and drives multiple proliferative pathways including activation of KIF11, induction of cyclinB and degradation of p53. KIF11 is also highly expressed in B-ALL and promotes cell cycle progression through enhanced spindle formation, while cyclinB promotes accelerated mitosis.

Through HES1 immunoprecipitation and MALDI-TOF peptide analysis, we identified KIF11 as a potential HES1 interacting protein. Low flow size exclusion chromatography revealed that HES1 and KIF11 co-migrate in high molecular weight complexes. Reciprocal immunoprecipitation (IP) with HES1 and KIF11 antibodies reveal a strong protein-protein interaction in B-ALL cells. PLK1 was also identified in these complexes, revealing a novel interaction of HES1, KIF11 and PLK1. Importantly, induction of Notch signaling via HES1 in B-ALL decreases both PLK1 and KIF11 levels, associated with G1 cell cycle arrest. Furthermore, HES1 expression decreased cyclinB protein levels, but not cyclin A/E, consistent with inhibition of PLK1. Finally, in the same model we observe increased p53 and p21^WAF1 protein levels, also contributing to growth arrest.

These data reveal that multiple mechanisms of Notch/HES1-mediated growth arrest may occur via PLK1 in B-ALL. The novel interaction of HES1 with PLK1 and KIF11 and resulting effects on KIF11, cyclinB and p53/p21 pathways may represent important mechanisms of Notch-mediated growth inhibition in B-ALL.