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The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients

Leukemia volume 28, pages 451–455 (2014)Cite this article

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To define that the Axl 3′-UTR is a functional target of miR-34a, we first performed in vitro luciferase reporter gene assays using the reporter construct containing the entire Axl 3′-UTR and increasing amounts of miR-34a mimic or sc-miR in HEK293 (human embryonic kidney 293) cells. A dose-dependent reduction of the luciferase activity was observed when the reporter gene and miR-34a mimic were co-transfected (Figure 1b, left panel). As expected, miR-34a did not show any effect on the pMIR-luc-Axl 3′-UTR (−110) reporter gene lacking the miR-34a-binding site (Figure 1b, right panel). We also found that co-transfection of miR-34a mimic with a plasmid DNA expressing the full-length Axl gene reduced Axl expression in a dose-dependent manner (Figure 1c). In addition, enforced introduction of miR-34a mimic in primary CLL B cells reduced endogenous level of Axl (Figure 1d). Taken together, these results suggest that miR-34a targets the Axl 3′-UTR and reduces Axl protein level. During progression of our study,3 two independent groups of investigators have reported a similar complementary binding site for miR-34a in the Axl 3′-UTR;4, 5 however, their findings were limited to established cell lines such that any clinical correlation was less obvious.

These initial findings became of more clinical interest as miR-34a, a direct target of p53, is reported to be associated with the adverse outcome in CLL patients.6, 7, 8 Therefore, we interrogated whether enforced activation of p53 could reduce Axl expression in primary CLL B cells using a DNA-damaging agent, doxorubicin. Results demonstrated accumulation of p53 protein in doxorubicin-treated CLL B cells from all the CLL patients tested (n=14) who had various chromosomal abnormalities detected by fluorescence in situ hybridization (FISH), albeit at variable degrees (Figure 1e). We noted that CLL B cells with deletion of 17p13 or 11q23 (11q23 defects: P6, P7; 17p13 defects: P8, P9) after doxorubicin exposure, accumulated lower levels of p53 and p21, the latter a direct target of p53, relative to the CLL clones with wild-type p53 or the ATM gene, an upstream regulator of p53 function (Figure 1e). Consistent with p53 activation, substantial reduction of Axl expression on doxorubicin-treated CLL B cells was noted including the CLL clones with heterologous deletion of the p53 or ATM gene when compared with the untreated cells (Figure 1f). Next, to establish that it was the increased expression of miR-34a, which reduced Axl expression in doxorubicin-exposed CLL B cells, we measured the levels of mature miR-34a in these CLL B cells. Of note, CLL B cells do not express other members of the miR-34 family: miR-34b/c,8 which recognize the same target binding sequence as does miR-34a.2 An increase of mature miR-34a at variable levels was detected in doxorubicin-treated CLL B cells from different patients (Figure 1g). Interestingly, leukemic B cells with 11q23/17p13 defects also showed variable degrees of miR-34a upregulation in response to doxorubicin (Figure 1g).

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Acknowledgements

This work was supported, in part, by a Mayo Clinic Hematology Research Award 2012, Eagles Cancer Research Award and research fund from National Cancer Institute CA170006-01A1 to AKG and research fund from National Cancer Institute CA95241 to NEK. We also acknowledge Tolero for providing us with the Axl inhibitor SGI-7079 and excellent secretarial help from Ms Tammy Hughes.

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  1. J Boysen and S Sinha: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Hematology, Mayo Clinic, Rochester, MN, USA

    J Boysen, S Sinha, T Price-Troska, D Viswanatha, T D Shanafelt, N E Kay & A K Ghosh

  2. Tolero Pharmaceuticals Inc., Salt Lake City, UT, USA

    S L Warner & D J Bearss

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  1. J Boysen
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Correspondence to A K Ghosh.

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Boysen, J., Sinha, S., Price-Troska, T. et al. The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients. Leukemia 28, 451–455 (2014). https://doi.org/10.1038/leu.2013.298

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