ARRB1 facilitated NOTCH1 ubiquitination and degradation through interactions with NOTCH1 and DTX1. (BUTR) and inhibits its expression in T-ALL. Furthermore, overexpression of the ARRB1-derived miR-223 sponge suppressed T-ALL cell proliferation and induce apoptosis. Collectively, these results demonstrate that ARRB1 acts as a tumor suppressor in T-ALL by promoting NOTCH1 degradation, which is usually inhibited by elevated miR-223, suggesting that ARRB1 may serve as a valid drug target in the development of novel T-ALL therapeutics. Introduction Clinically characterized by high white blood cell counts, hepatosplenomegaly, an increased risk of central nervous system infiltration and high relapse rates, T-ALL is usually associated with inferior prognosis. Although the success rates for acute lymphoblastic leukemia (ALL) treatment have markedly improved, the 5 12 months event-free survival rate Galidesivir hydrochloride of T-ALL is usually approximately 80%, significantly lower than that of B cell acute lymphoblastic leukemia (B-ALL; ref. 1,2). Thus, there is an urgent clinical need to develop novel and efficacious therapeutics for T-ALL, which can be greatly facilitated by understanding the molecular mechanisms underlying leukemogenesis. Galidesivir hydrochloride The constitutive activation of NOTCH1 is the most prominent oncogenic pathway, presenting in nearly 70% of T-ALL patients (3,4). The NOTCH1 pathway is usually activated by the ligand-mediated proteolytic release and translocation of intracellular NOTCH1 (ICN1) to the nucleus, where it regulates the expression of target genes. NOTCH1 deprivation during hematopoiesis leads to an absence of T cells in the thymus (5). In contrast, the overexpression of ICN1 in hematopoietic stem cells (HSCs) induces extrathymic T-cell development (6,7), even T-ALL transformation (8). Galidesivir hydrochloride Two categories of NOTCH1 mutations are typically identified in T-ALL patients. The more common NOTCH1 mutations (40C45% of tumors) occur in the heterodimerization domain name (HD; ref. 3,4), while the other type of mutations (30% of tumors) occur in the C-terminal PEST domain (9).?Nonetheless, NOTCH1 mutations alone are not sufficient to drive the development of full-blown leukemogenesis, suggesting that additional genetic and/or epigenetic alterations may be required for T-ALL development and progression (10). As members of the -arrestin (ARRB) protein family, -arrestin1 (ARRB1) was originally identified as a molecule involved in the desensitization and endocytosis of G protein coupled receptors (GPCRs; ref. 11C13). Although the functions of these proteins are not completely comprehended, ARRBs are versatile and multifunctional adapter proteins that regulate a diverse array of cellular functions (14C18). ARRB1 also serves as an E3 ligase adaptor for its substrates to Galidesivir hydrochloride mediate ubiquitination (19C23). We previously showed that ARRB1 is usually abundantly expressed in leukemia-initiating cells and can sustain the renewal capacity and senescence of cells, leading to the growth of B cells to form B-ALL (24,25). However, little is known regarding the potential role of ARRB1 in T-ALL development and progression. In this study, we investigated the role of ARRB1 in T-ALL progression. We showed that ARRB1 inhibits the progression of T-ALL cells by serving as a scaffold and interacting with NOTCH1 and DTX1 to facilitate the ubiquitination and degradation of NOTCH1. Moreover, the exogenous expression of miR-223 was shown to lead to a significant decrease in ARRB1 expression in T-ALL cells, which can be rescued by an miR-223 sponge. The data suggest that ARRB1 may serve as a Galidesivir hydrochloride valid drug target for the development of novel and efficacious therapeutics for T-ALL treatment. Materials and Methods Cell culture and chemicals HEK-293T and human T-ALL cell MAP3K11 lines, including Molt4, CCRF-CEM, and Sup-T1 were obtained from ATCC (Manassas, VA). Jurkat, Cutll1 and Molt3 T-ALL lines were kindly provided by Dr. Panagiotis Ntziachristos (26). All T-ALL cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Invitrogen, USA), L-glutamine and penicillin/streptomycin, while HEK293T cells were maintained in complete DMEM. Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). All cell lines were obtained more than 6 months prior to experiments and were passaged for less than 3 months after thawing. All cell lines were cultured according to the manufacturers instructions and confirmed as Mycoplasma unfavorable by PCR methods. Cellular experiments were performed within 20 passages after thawing. The information of the T-ALL lines is usually provided in Supplementary Table 1. T-ALL clinical samples The enrollment and human subject protection plans for the T-ALL patients involved in this study were approved by the Ethics Committee of Chongqing Medical University, Chongqing, China. Prior to the collection and use of the clinical samples, patients and their guardians were provided with detailed information about the benefits and risks of the study. The written informed consent forms were signed by the guardians during their.
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