ARRB1-Promoted NOTCH1 Degradation Is Suppressed by OncomiR miR-223 in T-cell Acute Lymphoblastic Leukemia

Clinically characterized by high white blood cell counts, hepatosplenomegaly, an increased risk of central nervous system infiltration and high relapse rates, T-cell acute lymphoblastic leukemia (T-ALL) is associated with inferior prognosis. Although the success rates for ALL treatment have markedly improved, the 5-year event-free survival rate of T-ALL is approximately 80%, significantly lower than that of B-cell ALL (B-ALL; refs. 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.

The constitutive activation of NOTCH1 is the most prominent oncogenic pathway, presenting in nearly 70% of patients with T-ALL (3, 4). The NOTCH1 pathway is 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 (HSC) induces extrathymic T-cell development (6, 7), even T-ALL transformation (8). Two categories of NOTCH1 mutations are typically identified in patients with T-ALL. The more common NOTCH1 mutations (40%–45% of tumors) occur in the heterodimerization domain (3, 4), whereas 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 β-arrestin (ARRB) protein family, β-arrestin1 (ARRB1) was originally identified as a molecule involved in the desensitization and endocytosis of G protein–coupled receptors (GPCR; refs. 11–13). Although the functions of these proteins are not completely understood, ARRBs are versatile and multifunctional adapter proteins that regulate a diverse array of cellular functions (14–18). ARRB1 also serves as an E3 ligase adaptor for its substrates to mediate ubiquitination (19–23). We previously showed that ARRB1 is abundantly expressed in leukemia-initiating cells and can sustain the renewal capacity and senescence of cells, leading to the expansion 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 valid drug target for the development of novel and efficacious therapeutics for T-ALL treatment.

HEK293T and human T-ALL cell lines, including Molt4, CCRF-CEM, and Sup-T1 were obtained from ATCC. Jurkat, Cutll1, and Molt3 T-ALL lines were kindly provided by Dr. Panagiotis Ntziachristos (Northwestern University, Evanston, IL; ref. 26). All T-ALL cell lines were maintained in RPMI1640 supplemented with 10% FBS (Invitrogen), l-glutamine, and penicillin/streptomycin, whereas HEK293T cells were maintained in complete DMEM. Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich or Thermo Fisher Scientific. 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 manufacturer's instructions and confirmed as Mycoplasma negative by PCR methods. Cellular experiments were performed within 20 passages after thawing. The information of the T-ALL lines is provided in Supplementary Table S1.

The enrollment and human subject protection plans for the patients with T-ALL 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 hospitalization at the Children's Hospital of Chongqing Medical University (Chongqing, China) according to the Declaration of Helsinki. The completed consent forms were kept on file. All essential patient identifiers were removed to protect the patients' privacy. Primary leukemic cells were isolated from bone marrow aspirates of freshly diagnosed patients with T-ALL prior to chemotherapy by gradient centrifugation. A normal peripheral T-cell population was enriched from age-matched healthy donors using an EasySep Human T-Cell Isolation Kit (Stem Cell Tech). The accession number for the RNA-seq data of patients with T-ALL is GEO Submission GSE141140. The clinical characteristics and genetic alterations of the used samples are listed in Supplementary Table S2.

The use and care of the experimental animals was approved by the Ethics Committee of Chongqing Medical University. Briefly, young NOD/SCID mice (6–8 weeks old, male), which were pretreated with anti-CD122 (kindly provided by Dr. Dengli Hong; ref. 27), were subjected to a sublethal dose irradiation (300 cGy) at the Radiation Facility of the Army Medical University, Chongqing, China. Subsequently, 3 × 10⁵ T-ALL cells per mouse were tail-vein injected into the irradiated NOD/SCID mice. The injected mice were observed for survival for up to 60 days after injection. For optical imaging, mice were intraperitoneally injected with d-luciferin sodium salt (Gold Biotechnology) at 100 mg/kg in 0.1 mL of PBS. Pseudo-images were obtained by superimposing the emitted light over grayscale images of the mice.

All quantitative experiments were performed in triplicate and/or repeated in three independent experiments. The data are presented as the means ± SD. Statistical significances between the treatments and control groups were determined by one-way ANOVA and Student t test. A value of P < 0.05 was considered significant.

To elucidate the function of ARRB1 in T-ALL cells, we constructed retroviral vectors to overexpress ARRB1 (β1) as well as two shRNAs targeting ARRB1, sh-ARRB1 #1 and #2 (sh-1 and sh-2), which coexpress GFP and firefly luciferase. After transducing the T-ALL cell lines with the retroviral vectors, we injected them into the immunodeficient mice to assess the function of ARRB1 in T-ALL cells in vivo. Surprisingly, unlike the previously reported tumor-promoting activity of ARRB1 in B-ALL cells (24, 25), the bioluminescence imaging results showed a slight tumor burden in mice grafted with ARRB1-overexpressing T-ALL cells compared with that observed in the scrambled vector–expressing T-ALL cells. Moreover, we also observed greater organ infiltration in the mice xenografted with ARRB1 knockdown T-ALL cells (Fig. 1A). Histologic images consistently showed a significantly lower tumor burden in the organs of mice injected with ARRB1-overexpressing T-ALL cells comparing with the control group, and these mice also exhibited a higher survival rate (Fig. 1A–D; Supplementary Figs. S1A and S1B). In contrast, silencing ARRB1 in T-ALL cells led to a significant accumulation of tumor cells in mouse organs and a decreased survival rate compared with that observed for the parental control group (Fig. 1A–D; Supplementary Figs. S1A and S1B). The CCK8 assay results demonstrated that ARRB1 overexpression led to a significant decrease in cell proliferative activity in all tested T-ALL lines (Fig. 1E; Supplementary Fig. S1C). Consequently, although the rate of GFP⁺ cells observed in the parental Molt3 cells was stable, GFP expression in the Molt3 cells overexpressing ARRB1 was gradually lost over subsequent passages (Supplementary Fig. S1D). The cell cycle and apoptosis assay results indicated that ARRB1 overexpression in T-ALL cells arrested the cells in the G₀ phase (Fig. 1F) and induced apparent apoptosis (Fig. 1G and H; Supplementary Figs. S1E and S1F). Collectively, these data indicate the ARRB1 suppresses T-ALL tumor progression in vivo and in vitro.

Considering that ARRB1 may serves as an important tumor suppressor of T-ALL progression, we next assessed the complete transcriptomic profiles of a panel of clinical primary T-ALL samples (n = 13) and peripheral T cells from healthy donors (n = 4) by performing RNA-seq analysis (Supplementary Table S2). Although numerous genes, including cancer-associated pathways such as NOTCH1, PI3K, RAS, WNT, and TP53, exhibited aberrant expression patterns in T-ALL cells, we observed the abnormal expression of several T-ALL–specific oncogenes, such as TAL1, LYL1, and LMO1/2 (Fig. 2A; Supplementary Figs. S2A and S2B; Supplementary Table S3). Furthermore, the clustering analysis results indicated an overall trend of a lower level of ARRB1 expression in the T-ALL samples than was observed in the T-cell controls (Fig. 2A).

To confirm the RNA-seq results, we analyzed the GEO databases and discovered that the expression of ARRB1 was generally downregulated in T-ALL cells compared with that observed in normal bone marrow cells (Fig. 2B). Furthermore, we enrolled additional patients with T-ALL (n = 17) and analyzed ARRB1 expression by qPCR analysis. The results indicated that ARRB1 expression was significantly lower in 13 of the 17 analyzed T-ALL samples compared with that observed in the normal T-cell controls (Fig. 2C; Supplementary Fig. S2C). Accordingly, the ARRB1 protein levels were lower than those observed in the control samples (Fig. 2D; Supplementary Fig. S2D). Hierarchical clustering analysis failed to distinguish between the ARRB1 expression levels and the patients bearing NOTCH1/FBXW7 mutations (Supplementary Table S2; Supplementary Figs. S2E–S2G). Taken together, these results strongly suggest that ARRB1 expression is downregulated in T-ALL.

To elucidate the functional role of ARRB1-induced suppression of T-ALL progression, we examined the transcriptomic changes of individual Jurkat cells with altered ARRB1 expression through an RNA-seq analysis. The expression heatmap shows the genes that were positively and negatively associated with ARRB1 (Fig. 3A). Genes there were positively associated with ARRB1 included those involved in the response to GPCR desensitization and signal conduction, such as PPARA, GRK4, and DNM1. Interestingly, genes that were negatively associated with ARRB1 included several T-ALL–related oncogenes, such as TAL1 and JAK2, as well as the NOTCH1 target genes HES1 and HES2 (Fig. 3A; Supplementary Table S4).

We verified the RNA-seq data in three T-ALL lines (Jurkat, Molt4, and Cutll1) by qPCR (Fig. 3B–D) and observed that HES1 and HES2 were consistently and negatively regulated by ARRB1 overexpression in the three tested lines, whereas silencing ARRB1 led to increased HES1 and HES2 expression in T-ALL cells (Fig. 3B–D). The inverse relationship between ARRB1 expression and that of HES1 and TAL1 was confirmed at the protein level (Fig. 3E). Collectively, these results strongly suggest that ARRB1 may exert its tumor-suppressive function by downregulating T-ALL–specific oncogenic events, especially those associated with the NOTCH1 signaling pathway.

To further assess how ARRB1 regulates NOTCH1 signaling in T-ALL cells, we first tested whether ARRB1 expression affects NOTCH1 expression and observed that either the overexpression or silencing ARRB1 expression in Jurkat and Molt4 cells did not significantly impact the levels of NOTCH1 mRNA (Fig. 4A) or ICN1 protein (Fig. 4B; Supplementary Fig. S3A) under conventional assay conditions. As ARRB1 was reported to be involved in the NOTCH1 cleavage and the release of the ICN1 domain by participating in the assembly of the γ-secretase complex (28), we tested the effect of ARRB1 on the formation of ICN1 in T-ALL cells using a γ-secretase inhibitor and showed that the ICN1 levels were not significantly affected by the ARRB1 expression status (Supplementary Fig. S3B).

ARRB1 was reported to mediate the interaction between E3 ubiquitin ligases DTX1 or NEDD4 and their substrate NOTCH1 to promote the ubiquitination and degradation of NOTCH1 in Drosophila and mice (21, 23). To more rigorously assess the dynamic status of ICN1 degradation, we blocked protein synthesis using cycloheximide. The results showed that ICN1 protein levels significantly decreased 2 hours after treatment in T-ALL cells overexpressing ARRB1 (β1) compared with that observed in the control cells, whereas silencing ARRB1 (using sh-1 and sh-2) led to relatively stable ICN1 protein levels in both cell lines (Fig. 4C; Supplementary Fig. S3C), suggesting that ARRB1 overexpression may promote the ICN1 degradation. To further confirm the effect of ARRB1 on ICN1 degradation, we additionally blocked protein degradation using the proteasome inhibitor MG-132, after stimulating cells with cycloheximide. We observed that the ARRB1-mediated degradation of ICN1 was diminished and that the ICN1 protein level became similar to that of the control and ARRB1-silenced groups at the tested time points (Fig. 4D). Thus, these data demonstrate that ARRB1 promotes the degradation of ICN1 in T-ALL cells.

To investigate whether ARRB1 regulates NOTCH1 degradation through its ubiquitination, we performed ICN1 pull-down assays using ARRB1-overexpressing or ARRB1-silenced T-ALL cells and Western blotted with an ubiquitin antibody. We found a significant increase in ICN1 ubiquitination in all T-ALL cells overexpressing ARRB1 compared with that observed in the control or ARRB1 knockdown groups (Fig. 5A; Supplementary Fig. S3D). Furthermore, immunofluorescence staining analysis revealed that ARRB1 and NOTCH1 colocalized in the cytoplasm of Jurkat cells after EDTA stimulation (Fig. 5B; ref. 29).

The results of coimmunoprecipitation experiments showed that ARRB1 directly interacted with ICN1 in Jurkat cells, suggesting that ARRB1 may directly regulate the ubiquitination of ICN1 protein (Fig. 5C). After screening candidate E3 ligases of ICN1, we observed that DTX1, which can catalyze the ubiquitination and subsequent degradation of ICN1 in Drosophila and mammalian cells (30), but not FBXW7, NEDD4, or ITCH was able to bind to ARRB1 in Jurkat cells (Fig. 5D; Supplementary Figs. S4A–S4C).

To further investigate the functional role of the ARRB1–DTX1 complex in promoting the ubiquitination of ICN1, we transfected HEK293T cells with exogenous His-ICN1 alone or in combination with HA-ARRB1 and/or Flag-DTX1. The coimmunoprecipitation results showed that neither ARRB1 nor DTX1 alone was sufficient to catalyze the ubiquitination of ICN1 protein, whereas in combination they were able to induce a high level of ICN1 ubiquitination (Fig. 5E). Furthermore, by silencing either ARRB1 or DTX1 we demonstrated that ARRB1 and DTX1 acted synergistically to promote the ICN1 ubiquitination in HEK293T cells (Fig. 5F). Taken together, these results demonstrate that ARRB1 can synergize with DTX1 to catalyze the ubiquitination of ICN1 for targeted proteasomal degradation.

As a NOTCH1 target gene, DTX1 was shown to redirect lymphoid progenitor cells to B cells by antagonizing NOTCH1 signaling during lymphogenesis (31, 32), as well as serving as a suppressor by mediating the ubiquitination and subsequent degradation of NOTCH1 in sarcoma cells (33). However, it is not clear whether DTX1 plays a role in T-ALL. By mining the GEO database, we observed that DTX1 expression was not significantly affected in T-ALL (Supplementary Fig. S5A). Consistent with these results, we did not observe any differences in DTX1 expression between our clinical samples and the normal controls (Supplementary Fig. S5B). We further knocked down DTX1 expression in Jurkat cells or overexpressed ARRB1 (Supplementary Fig. S5C and S5D) and observed that silencing the DTX1 expression in ARRB1 overexpression T-ALL cells could overcome the ARRB1-mediated growth inhibition of T-ALL cells (Supplementary Fig. S5E), suggesting that the ARRB1-mediated inhibition of NOTCH1 and T-ALL depends on DTX1.

The next question we asked is why ARRB1 is poorly expressed in T-ALL. Although ARRB1 expression can be regulated at the genomic level, an alternative possibility is that ARRB1 may be epigenetically regulated, as it was reported that epigenetic aberrations are prevalent in T-ALL (4, 26, 34–36). Thus, we speculated that ARRB1 may be downregulated by noncoding RNAs, especially miRNAs, which can reduce the expression of their target genes by promoting mRNA degradation or inhibiting translation (37). Our RNA-seq results for the T-ALL clinical samples indicated that miR-223 was the most highly expressed miRNA in the analyzed T-ALL samples (Fig. 6A). By searching several miRNA target site prediction bioinformatic programs, we further showed that miR-223-3p can target the 3′-UTR of ARRB1 (referred to hereafter as BUTR; Fig. 6A). Interestingly, miR-223 is considered a hematopoietic-specific oncomiR.

To determine whether miR-223 directly targets ARRB1, we constructed a reporter by subcloning the 402-bp BUTR fragment into the 3′ end of the GLuc coding region, referred to hereafter as GLuc-BUTR (Fig. 6A and B). As a control, we scrambled the ARRB1-binding site and made a control reporter (GLuc-SCR; Fig. 6A and B). We observed that miR-223 was able to inhibit GLuc-BUTR reporter activity, whereas this inhibition was not observed for the control reporter (Fig. 6B). Furthermore, we established a Jurkat line that stably overexpresses miR-223 and showed that the expression of ARRB1 and other known miR-223 target genes was effectively downregulated in these cells compared with those transfected with the miR-223 scrambled (scr) sequence control (Fig. 6C), which was confirmed in other T-ALL lines and HEK293T cells (Fig. 6D). In contrast, the expression of a miR-223 sponge (AM223) in Jurkat cells expressing miR-223 was shown to rescue the expression of ARRB1 and other target genes (Fig. 6E), suggesting that miR-223 may target ARRB1 with high specificity. We further confirmed the inhibitory effect of miR-223 on ARRB1 expression at the protein level in T-ALL cells (Fig. 6F). Taken together, these results strongly suggest that miR-223 may potently and specifically target ARRB1 in T-ALL cells.

We next tested whether the ARRB1 3′-UTR containing the miR-223 binding site (BUTR) could act as a miR-223 sponge and negatively regulate the proliferation of T-ALL cells by constructing retroviral (RV) vectors that coexpress mRFP and BUTR or the scrambled SCR control (Fig. 7A). Using these vectors, we observed that the overexpression of BUTR but not the control SCR significantly inhibited Jurkat cell proliferation, which was rescued by the simultaneous expression of miR-223 (Fig. 7B).

We further examined if overexpression of the miR-223 sponge BUTR would cause apoptosis in T-ALL cells and showed that BUTR overexpression in Jurkat and CCRF-CEM cells induced apparent apoptosis, as indicated by the presence of cleaved caspases 3 (Fig. 7C; Supplementary Fig. S6). Consistent with results of the apoptosis assay, the overexpression of BUTR in Jurkat cells effectively arrested cells in the G₀–G₁ phases and reduced the cell numbers in the G₂–S phases (Fig. 7D).

We further examined the specificity of the ARRB1-derived anti–miR-223 sponge and observed that the overexpression of BUTR effectively rescued ARRB1 expression, and to lesser extent FBXW7 expression, without significant rescue of the additional miR-223 target genes RHOB and HLF (Fig. 7E). The Western blotting results also revealed that BUTR rescued the ARRB1 protein levels while also suppressing the level of ICN1 and its target gene DTX1 (Fig. 7F). Collectively, these results demonstrate that the exogenous expression of the miR-223 sponge BUTR may be incompatible with the survival of T-ALL cells. These results also suggest that ARRB1-based antagonism of miR-223 may be further explored as a novel T-ALL therapeutic.

Although β-arrestins (ARRB) were originally identified for their ability to block agonist coupling to GPCRs, increasing evidence suggests that ARRBs may have diverse biological functions independent of G protein activation (11–13). By serving as multiprotein scaffolds to bring components of various signaling pathways into close proximity, ARRBs have been shown to participate in signaling activities transduced by ERK, JNK, p38-MAPK, AKT, PI3 kinase, and RhoA (14–18, 38, 39). ARRBs can be translocated to the nucleus and regulate gene expression (12). Furthermore, ARRB1 can serve as an E3 ligase adaptor for its substrates, such as NOTCH1, to mediate ubiquitination through its interaction with DTX1 (19–22). Nonetheless, the diverse functions mediated by ARRBs remain incompletely understood.

We previously showed that the abundant expression of ARRB1 in leukemia-initiating cells can sustain the renewal capacity and senescence of cells, leading to the expansion of B cells to form B-ALL (24, 25). Collectively, the results of the vast majority of recent studies indicate that ARRB-dependent signaling contributes to cancer phenotypes in specific malignancies. In this study, we uncovered a tumor-suppressing role for ARRB1 in T-ALL, which was supported by the expression data from clinical samples and the results of in vitro and in vivo analyses. Furthermore, we identified a novel underlying mechanism through which ARRB1 expression is targeted by the oncomiR miR-223 in T-ALL cells.

Consistent with our findings, the results of a recent study by Ferrandino and colleagues (40) also suggested a tumor-suppressing role of ARRB1 during T-ALL initiation in NOTCH3 transgenic mice, although through a different mechanism. In the transgenic mice, NOTCH3 upregulates the CXCR4 receptor on the surfaces of CD4 and CD8 double-positive thymocytes by catalyzing the phosphorylation of ARRB1 and inhibiting the internalization of CXCR4. Consequently, NOTCH3 and CXCR4 positive thymocytes escape the thymus and ultimately colonize bone marrow to degenerate into T-ALL. Taken together, these results and our current findings reveal a previously unrecognized tumor-suppression function of ARRB1 in T-ALL, indicating that further investigation is thus highly warranted.

miRNAs are a class of small noncoding RNAs consisting of 18 to 25 nucleotides. By pairing the seed sequence with their target mRNAs, miRNAs regulate target gene expression and tune biological processes (41). The dysregulation of miRNAs contributes to the initiation and maintenance of cancer (42). As an oncomiR, miR-223 has been reported to play important roles in inflammation, infection, and cancer development (43–45). We observed that miR-223 was abundantly expressed in most T-ALL patient samples, suggesting that it has a potentially important role in T-ALL. Interestingly, ectopic miR-223 was previously shown to cooperate with ICN1 to initiate the leukemogenesis of a T lineage in a xenograft mouse model (36). More recently, miR-223 was identified as the most dynamically regulated miRNA by the T-ALL–specific oncogenic transcription factor TAL1 (46). Aberrant miR-223 upregulation by TAL1 is crucial for the optimal growth of TAL1-positive T-ALL cells, and miR-223 overexpression led to a marked downregulation of FBXW7 protein expression, suggesting that TAL1-mediated upregulation of miR-223 may promote the malignant T-ALL phenotype by repressing FBXW7 tumor suppressor (46). Interestingly, another recent study reported that the promoter region of miR-223 contains a binding site for RBPJ, a coactivator of NOTCH1, and that NOTCH1-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines (47), suggesting there may be a positive feedback regulatory circuitry between NOTCH and miR-223. In this study, we further demonstrated that ARRB1 can promote NOTCH1 degradation in T-ALL, which is effectively inhibited by miR-223.

In summary, in this study, we investigated whether ARRB1 plays a role in T-ALL development and/or progression. We conducted an RNA-seq analysis of T-ALL clinical samples and observed that ARRB1 was poorly expressed in T-ALL clinical samples and cell lines. Exogenous ARRB1 expression inhibited T-ALL proliferation and drastically improved the survival rate of xenograft animals. In addition, we observed that the tumor suppression activity was at least in part attributable to ARRB1-mediated NOTCH1 degradation. Further analysis revealed that the oncomiR miR-223 targets the 3′-UTR of ARRB1 (aka, BUTR) to inhibit ARRB1 expression in T-ALL. Furthermore, we demonstrated that the overexpression of the ARRB1-derived miR-223 sponge BUTR was incompatible with cell proliferation and induced apoptosis in T-ALL cells. Thus, our results demonstrate a tumor-suppressing role for ARRB1 in T-ALL, which is effectively antagonized by miR-223. It is conceivable that miR-223 may serve as a valid drug target for the development of novel and efficacious therapeutics for T-ALL treatment.

No potential conflicts of interest were disclosed.

Conception and design: T.-C. He, L. Zou

Development of methodology: T.-C. He

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Shu, Y. Wang, W.-Q. Lv, D.-Y. Peng, H. Zhang, G.-J. Jiang, B.-J. Yang, S. Liu, J. Zhang, Y.-H. Chen, S. Tang, J.-T. Yuan, W. Guo, G. Fu, X.-K. Qi, H.-Y. Liu, C. Yang, L.-H. Zhang, F.-J. Liu, J. Yu, P.-H. Zhang, B. Qu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Shu, D.-Y. Peng, S. Liu, Z.-D. Liu, T.-C. He, L. Zou

Writing, review, and/or revision of the manuscript: Y. Shu, J. Li, S. Liu, H. Zhao, T.-C. He, L. Zou

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Liu, S. Tang, K.-X. Wan, G. Fu, Z.-D. Liu

Study supervision: T.-C. He, L. Zou

We are greatly indebted to the patients with T-ALL and their families for their support and willingness to participate in the study. We are grateful for Dr. Gang Pei of Institute of Biochemistry and Cell Biochemistry, Shanghai Institute of Biomedical Sciences, Shanghai, China, for his generous provision of ARBB1-related constructs and scientific advice during the course of investigation. We thank Dr. Panagiotis Ntziachristos of Northwestern University for the provision of Jurkat and Cutll1 cell lines. We also thank Dr. Qing Lu of the University of Arizona for the critical review of the data and manuscript. The reported work was supported in part by research grants from the National Natural Science Foundation of China (#81373444 and #81570142 to L. Zou), the NIH (CA226303 to T.-C. He), and the National Key Research and Development Program of China (#2016YFA0101300 to L. Zou). Y. Shu was a recipient of the Predoctorate Fellowship from the China Scholarship Council. This project was also supported in part by The University of Chicago Cancer Center Support Grant (P30CA014599) and the National Center for Advancing Translational Sciences of the NIH through grant number UL1 TR000430. T.-C. He was also supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund. Funding sources were not involved in the study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the manuscript for publication.

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