Potential therapeutic role of antagomiR17 for the treatment of chronic lymphocytic leukemia

Recently it was reported that microRNA from the miR-17 ~ 92 family may have a key role in chronic lymphocytic leukemia (CLL). Here, we designed specific oligonucleotides to target endogenous miR-17 (antagomiR17). In-vitro administration of antagomiR17 effectively reduced miR-17 expression and the proliferation of CLL-like MEC-1 cells. When injected in-vivo in tumor generated by the MEC-1 cells in SCID mice, antagomiR17 dramatically reduced tumor growth and significantly increase survival. Altogether, our results provide the rationale for the use of antagomiR17 as a novel potential therapeutic tool in CLL and in other lymphoproliferative disorders where miR-17 has a driver role in tumor progression.

section_content: We have recently reported that microRNA from the miR-17 ~92 family may be responsible for the increased proliferation/survival in chronic lymphocytic leukemia (CLL) cells expressing unmutated (UM) IGHV genes and with high level of ZAP-70 [1]. In particular, the enforced expression of miR-17 reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected CLL cells from apoptosis [1]. Here, we provide evidences that the abrogation of miR-17 expression by a specific antagomiR is sufficient to inhibit leukemic growth and progression both in-vitro and in-vivo. 

Peripheral blood samples from CLL patients were obtained in accordance with local Institutional Review Board requirements and declaration of Helsinki. CLL cell stimulation, microRNA and gene expression were performed as reported [1, 2]. MEC-1 CLL-like cell line was transfected with a molecule against miR-17 (hereafter antagomiR17), or scrambled control. In in-vivo experiments, tumors generated by MEC-1 cells into severe combined immunodeficiency (SCID) mice were treated with antagomiR17, scrambled control, or saline solution (see Additional file 1). 

The MEC-1 cell line expressed miR-17 levels comparable to those of CLL samples in which proliferation is triggered by CpG-ODN (Figure 1a ). In MEC-1 cells, antagomiR17 transfection significantly reduced miR-17 expression respect to scrambled control, both at day 2 (mean fold change 0. 84 ± 0. 06; P = 0. 049) and at day 4 (mean fold change 0. 48 ± 0. 14; P = 0. 021; Figure 1b ). Moreover, the TP53INP1, TRIM8 and ZBTB4 expression showed a significant up-regulation after antagomiR17 treatment both at transcript and protein levels (Figure 1c, d ). Finally, MEC-1 cells showed a significant reduction (P = 0. 033) of cell rate proliferation when transfected with antagomiR17 (Figure 1e ). Complementary experiments performed using sorting procedures after transfecting MEC-1 cells with a Cy3-labelled antag-omiR17 (Cy3-antagomiR17, Additional file 2: Figure S1a ) showed that the Cy3-antagomiR17 bright fraction presented a significant decrease in cell proliferation respect to the Cy3-antagomiR17 dim fraction at day 7 (P = 0. 008; Additional file 2: Figure S1b ). Notably, using a Cy3-labelled scrambled control no difference in MEC-1 cell proliferation was observed (Additional file 2: Figure S1c, d ). Altogether, these data demonstrated that antagomiR17 administration effectively reduced the expression of miR-17 and cell proliferation. Tumors generated by MEC-1 cells injected into SCID mice were treated three times (day 1-8-15) either with antagomiR17 or scrambled control. AntagomiR17 dramatically inhibited tumor growth; this effect, already relevant after the first week of therapy, was maintained till the end of the treatment (Figure 2a ) leading to complete regression of the mass in 1/5 (20%) of cases (not shown). Conversely, administration of the scrambled control resulted in a tumor growth kinetic superimposable to saline-treated tumors (Figure 2a ). Of note, a single injection of antagomiR17 was sufficient to significantly reduce tumor growth for at least two weeks after treatment (Additional file 2: Figure S1e ). Consistently, median overall survival (OS) of mice treated with antago-miR17 was significantly longer than median OS of mice treated with scrambled control (91 versus 52 days, respectively, P = 0. 0018) or saline solution (91 versus 51 days, respectively, P = 0. 0044) (Figure 2b ). Notably, none of the mice showed signs of toxicity. Altogether, these results demonstrate that in-vivo treatment with anta-gomiR17 significantly abolishes tumor growth and increases survival. 

Evidences reported here underline that miR-17 knockdown is sufficient to block CLL-like cells proliferation both in-vitro and in-vivo. Clinically, despite recent treatment advances, some CLL seem to be refractory to the new drugs [3] [4] [5]. In this context, antagomiR treatment may represent a commendable alternative, also considering recent antagomiR phase II trials [6] [7] [8]. This strategy could be extended to other lymphoproliferative disorders where miR-17 ~92 amplification and/or overexpression have a pathogenetic role [9, 10]. In conclusion, our results highlight the therapeutic potential of antagomiR17, providing the rationale for its use also in the context of specific target delivering systems (e. g. nanoparticles).
section_name: Findings

section_name: Acknowledgements

section_content: Supported in part by: Ministero della Salute (Ricerca Finalizzata I. R. C. C. S., "Alleanza Contro il Cancro"; Rete Nazionale Bio-Informatica Oncologica/RN-BIO ; Progetto Giovani Ricercatori n. GR-2011-02347441, n. GR-2009-1475467, n. GR-2008-1138053, Ministero della Salute, Rome, Italy ; Fondazione Internazionale di Ricerca in Medicina Sperimentale (FIRMS) ; Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy ; Ricerca Scientifica Applicata, Regione Friuli Venezia Giulia ("Linfonet" Project), Trieste, Italy; the Associazione Italiana Ricerca Cancro (AIRC), Grants n. IG-13227, MFAG-10327, Milan, Italy; " 5x1000 Intramural Program ", Centro di Riferimento Oncologico, Aviano, Italy.
section_name: Acknowledgements

section_name: Additional files

section_content: Additional file 1: Supplemental material and methods. 

Additional file 2: Figure S1. In-vitro control experiments.
section_name: Additional files

section_content: The authors declare no competing financial interests. 

Authors' contributions SD, PM performed research, and contributed to write the manuscript; TDA, NM, MDB, SC, AZ, ET performed research and in vivo experiments; GDP provided patients data; SZ, in vivo experiments; VG, RB designed the study and wrote the manuscript. All authors read and approved the final manuscript.
section_name: Competing interests

section_content: Sara Dereani and Paolo Macor contributed to this study as first authors. Valter Gattei and Riccardo Bomben equally contributed to this study as senior authors.
section_name: Authors' information