Abstract
RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia.
Highlights
RUNX1 belongs to a family of transcriptional regulators, called RUNX (1)
Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1–RUNX1T1
Among the RUNX1-interacting E3 ligases, we found that STUB1 promotes ubiquitination of RUNX1 and RUNX1–RUNX1T1, and thereby inhibits their activities
Summary
We first synthesized recombinant proteins of RUNX1 and its cofactor CBFB with the wheat cell-free system in N-terminal FLAG-tagged forms and N-terminal single biotinylated forms. RUNX1 protein in STUB1-depleted K562 cells showed a slower rate of degradation (Fig. 3C) These data suggest that STUB1 is an E3 ubiquitin ligase that regulates RUNX1 stability. STUB1 depletion using the CRISPR/Cas system in Kasumi-1 cells led to increased stability and expression of RUNX1–RUNX1T1 (Fig. 7, A and B) Together, these data suggest that STUB1 acts as an E3 ligase for RUNX1–RUNX1T1 to regulate its expression. Nucleus even in STUB1-overexpressing cells, indicating that intracellular localization of RUNX1–RUNX1T1 is not regulated by STUB1 (Fig. 8, A and B). We showed that STUB1 depletion in K562 cells led to increased stability and expression of RUNX1 protein, indicating the essential role of endogenous STUB1 to promote RUNX1 ubiquitination in vivo. Our data suggest that STUB1 induces RUNX1 ubiquitination, promotes its degradation as well as nuclear export, and inhibits transcriptional activity of RUNX1. The PROTAC-based therapy, linking STUB1 and RUNX1, can be a promising therapy for RUNX1–RUNX1T1 leukemia and potentially for other leukemias with RUNX1 dysregulation
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