Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth
Coralie Hoareau-Aveilla, Thibaud Valentin, Camille Daugrois, Cathy Quelen, Géraldine Mitou, Samuel Quentin, Jinsong Jia, Salvatore Spicuglia, Pierre Ferrier, Monica Ceccon, Sylvie Giuriato, Carlo Gambacorti-Passerini, Pierre Brousset, Laurence Lamant, Fabienne Meggetto
Coralie Hoareau-Aveilla, Thibaud Valentin, Camille Daugrois, Cathy Quelen, Géraldine Mitou, Samuel Quentin, Jinsong Jia, Salvatore Spicuglia, Pierre Ferrier, Monica Ceccon, Sylvie Giuriato, Carlo Gambacorti-Passerini, Pierre Brousset, Laurence Lamant, Fabienne Meggetto
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Research Article Oncology

Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth

Abstract

The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.

Authors

Coralie Hoareau-Aveilla, Thibaud Valentin, Camille Daugrois, Cathy Quelen, Géraldine Mitou, Samuel Quentin, Jinsong Jia, Salvatore Spicuglia, Pierre Ferrier, Monica Ceccon, Sylvie Giuriato, Carlo Gambacorti-Passerini, Pierre Brousset, Laurence Lamant, Fabienne Meggetto

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Figure 3

The miR-150 target MYB is downregulated upon NPM-ALK inhibition.

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The miR-150 target MYB is downregulated upon NPM-ALK inhibition. (A) MYB...
(A) MYB mRNA levels were measured by qPCR in WT (n = 6) or NPM-ALK–transgenic mice upon induction (NPM-ALK[+] ON, no doxycycline, n = 11) or repression (NPM-ALK[–] OFF, with doxycycline, n = 6) of the NPM-ALK transgene. S14 served as an internal control, and relative MYB expression was expressed as the 2–ΔΔCt relative to WT or NPM-ALK(–) OFF mice. (B) The expression of miR-150 and MYB was evaluated by qPCR in KARPAS-299 cells untreated (PBS, vehicle control) or treated with 500 nM crizotinib for 24, 48, and 72 hours. miR-150 levels were normalized to those of RNU24, and MYB levels were normalized to those of GAPDH; these were both normalized to untreated conditions. (C and D) KARPAS-299 cells were transfected with control siRNA (si-CTL) or si-ALK. (C) MYB and ALK mRNA levels were analyzed by qPCR. Results were normalized to GAPDH and then normalized to si-CTL. Data represent mean ± SEM. n = 3; *P < 0.05, **P < 0.001, and ***P < 0.0001; unpaired 2-tailed Student’s t test. (D) The protein levels of NPM-ALK, STAT3, p-STAT3 (form of STAT3 phosphorylated on tyrosine 705), MYB, and DNMT1 were assessed by Western blotting after knockdown of NPM-ALK. GAPDH served as an internal control to ensure equal loading.