BCL11B promotes T‐cell acute lymphoblastic leukaemia cell survival via the XRCC5/C11ORF21 axis

B-cell lymphoma/leukaemia 11B (BCL11B) is an essential transcriptional regulator of T cells and is engaged in regulating vital biological processes including T-cell development, proliferation, differentiation and survival.1, 2 Previous studies have shown that BCL11B exhibits abnormally high expression levels in cases of T-cell acute lymphoblastic leukaemia (T-ALL) and that its deletion can significantly induce apoptosis of T-ALL cells.3, 4 However, the molecular mechanism underlying this process remains unclear. Here, we identified X-ray repair cross complementing protein 5 (XRCC5) as a binding protein of BCL11B and XRCC5 may regulate BCL11B expression in both transcriptional and protein level to inhibit the apoptosis of T-ALL. In addition, we also found a new target gene of BCL11B namely Chromosome 11 Open Reading Frame 21 (C11ORF21), which also has an effect on the pathogenesis of T-ALL. Our study indicates that the XRCC5/BCL11B/C11ORF21 signalling pathway is a potential target in the treatment of T-ALL. In this study, immunoprecipitation and mass spectrometry (MS) were first used to screen for binding proteins of BCL11B. In three T-ALL cell lines, CCRF, JURKAT and MOLT4, proteins binding BCL11B were identified through immunoprecipitation followed by MS (Figure 1A). Interestingly, XRCC5 was repeatedly identified in the MS data of all the three T-ALL cell lines, and was confirmed in the CCRF and 293T cells (Figure 1B,C). This result demonstrated that XRCC5 has an interaction with BCL11B. XRCC5 was initially recognised for its role in mending double-stranded DNA breaks; henceforth, it was deemed to promote therapeutic resistance against cancerous cells, induced by DNA-damaging agents.5-7 We then found XRCC5 binds to the X1 region of BCL11B, which contains Leucine-zipper domain that is responsible for sequence specific DNA binding (Figure 1D), indicating XRCC5 may function as a co-transcription factor with BCL11B to modulate the downstream gene expression. In addition, we also found that XRCC5 was significantly up-regulated in primary T-ALL cells and positively correlated with BCL11B expression (Figure 2A–D). Moreover, both BCL11B and XRCC5 expressions were inhibited in CCRF and JURKAT treated with Doxorubicin or Vincristine (Figure 2E,F), suggesting that targeting the XRCC5/BCL11B signalling pathway holds promise for the diagnosis and therapeutic intervention of T-ALL. These results suggest that expression of XRCC5 correlated with expression of BCL11B and play important roles in regulating the survival of T-ALL cells. We have previously shown that inhibition of BCL11B expression could significantly induce apoptosis in T-ALL cells8; thus, we next explored the function of XRCC5 in the survival of T-ALL cells. XRCC5 was shown to be effectively knocked down by siRNA (Figure 3A,B). As expected, the knockdown of XRCC5 promotes the apoptosis of CCRF and JURKAT cells (Figure 3C). It is noteworthy that the combination of XRCC5 and BCL11B siRNA treatment significantly enhanced CCRF and JURKAT cell apoptosis compared to single-gene siRNA (Figure 3D). In addition, down-regulation of XRCC5 can inhibit the expression of BCL11B. In contrast, inhibition of BCL11B has no effect on XRCC5 (Figure S1), suggesting that XRCC5 is an upstream regulator of BCL11B. Taken together, these data confirmed the involvement of XRCC5 and BCL11B in T-ALL survival. To delve deeper into the mechanism of XRCC5/BCL11B regulating T-ALL cell survival, we took the intersection of the differential genes and the genes strongly related to BCL11B in the GSE13159 dataset, and differential genes in the CCRF-siBCL11B dataset, and then C11ORF21, the downstream gene of BCL11B was screened (Figure 4A,B). Subsequently, we analysed the expression of C11ORF21 in 40 samples of T-ALL patients from our clinical centre and the data from GEO database (GSE13159). In contrast to XRCC5 and BCL11B, C11ORF21 was significantly down-regulated in T-ALL, and there was an inverse correlation between the expression of C11ORF21 and that of BCL11B (Figure 4C–F). In addition, C11ORF21 expression was activated when T-ALL was treated with Doxorubicin or Vincristine (Figure 4G,H). These results suggest that BCL11B may directly inhibit the expression of C11ORF21 which may benefit T-ALL cell survival. Furthermore, we confirmed that down-regulating the expression of BCL11B in JURKAT cells activated the expression of C11ORF21 (Figure 4I,J). In order to determine whether BCL11B can directly transcriptionally regulate the expression of C11ORF21, we found multiple BCL11B binding sites in the C11ORF21 gene through CUT-tag combined sequencing technology (Figure 4K). Taken together, these findings suggest that C11ORF21 serves as a novel BCL11B target gene involved in the regulation of T-ALL cell survival. C11ORF21 is a novel gene situated within the human chromosome 11p15.5 locus, with potential implications in Beckwith–Wiedemann syndrome and cancers.9 By northern blotting, this gene was found to exhibit exclusive expression solely in the human heart. Demonstration of C11ORF21–EGFP fusion protein proved that the encoding of a 132-amino acid protein by C11ORF21, predominantly situated within the cytoplasm.9 Until now, the biological function of C11ORF21 has not been elucidated. Recently, Matsumoto et al. showed that C11ORF21 is a novel target gene of RUNX1, while the fusion protein RUNX1–ETO inhibited C11ORF21 expression in AML1–ETO leukaemia.10 These findings suggest that RUNX1 was recruited as co-factor to around all classes of target genes of BCL11B in the T-cell fate commitment.1 Thus, BCL11B and RUNX1 may bind together to repress the expression of C11ORF21 in T-ALL, therefore, targeting C11ORF21 might be a latent way to treat T-ALL. However, the co-regulation of RUNX1 and BCL11B remained to be explore in the future. Taken together, using immunoprecipitation, real-time quantitative polymerase chain reaction, siRNA and other methods, our work suggests that XRCC5 can bind to BCL11B, subsequently suppressing C11ORF21, and facilitating the survival of T-ALL cells (Figure S2). However, there are limitations in this study, which are needed to further investigation. First, the molecular mechanism underlying the regulation of BCL11B by XRCC5 is not yet fully elucidated. Second, although XRCC5 and C11ORF21 have been found to be dysregulation in T-ALL, their roles in vivo, along with their involvement in T-ALL development and treatment, remain to be extensively investigated. In conclusion, the XRCC5/BCL11B/C11ORF21 axis has a crucial role in the development of T-ALL and may serve as a promising candidate for therapeutic interventions against T-ALL. Xibao Yu, Yuchen Li and Pengyue Yang performed the experiments, wrote the paper and analysed the data. Yan Wang, Xuan Liu and Letong Cai helped analyse the data. Jing Lai, Yue Zhang and Xianfeng Zha diagnosed and treated the patients and collected clinical samples. Grzegorz Krzysztof Przybylski helped to edit the results and revise the manuscript. Yangqiu Li, Ling Xu and Xibao Yu designed the study and wrote the manuscript. All the authors read and approved the final manuscript. We acknowledge the GEO (https://www.ncbi.nlm.nih.gov/geo/) (GSE13159) for providing the datasets. This work was supported in part by the Intergovernmental International Cooperation on Scientific and Technological Innovation Project of Chinese Ministry of Science and Technology (no. 2017YFE0131600), the National Natural Science Foundation of China (nos. 82200167 and 82000108), the Guangdong Basic and Applied Basic Research Foundation (no. 2021A1515110140), the National Innovation and Entrepreneurship Training Program for Undergraduate (no. 202310559052) and the National Centre for Research and Development, Poland (no. WPC/BCL/2019). The authors declare they have no conflicts of interest. The study (no. 20220223) was approved by the Ethics Committee of the Affiliated Hospitals of Jinan University. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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