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01.12.2017 | Letter to the Editor | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Riccardo Masetti, Salvatore Nicola Bertuccio, Annalisa Astolfi, Francesca Chiarini, Annalisa Lonetti, Valentina Indio, Matilde De Luca, Jessica Bandini, Salvatore Serravalle, Monica Franzoni, Martina Pigazzi, Alberto Maria Martelli, Giuseppe Basso, Franco Locatelli, Andrea Pession
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0396-0) contains supplementary material, which is available to authorized users.
Abbreviations
AML
Acute myeloid leukemia
AURKA
Aurora A kinase
BMP
Bone morphogenic protein
ChIP
Chromatin immunoprecipitation
FAB
French-American-British
GLIS2
GLI-similar protein 2
HH
Hedgehog
PI
Propidium iodide
WST1
4-[3-(4 lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate)

Findings

Pediatric acute myeloid leukemia (AML) carrying CBFA2T3- GLIS2 fusion gene deserves particular interest, being associated with a grim prognosis in all the reports published so far [ 13]. The incidence of this aberration is 17 and 8% in pediatric non-Down syndrome acute megakaryoblastic and in pediatric cytogenetically normal AML, respectively [ 13]. The expression profile of CBFA2T3- GLIS2 is associated with upregulation of both Hedgehog (HH) and bone morphogenic protein (BMP) signaling [ 1, 4].
The protein GLIS2 shares a highly homologous zinc finger domain with members of the GLI proteins, the final effectors of classic Hedgehog pathway. GANT61 is a GLI inhibitor showing a potent effect on the inhibition of transcription activity of GLI proteins, blocking their binding to DNA [ 58]. Considering the high homology of the DNA-binding domain between GLIS2 and GLI family proteins, we hypothesized that GANT61 might be used to specifically target the CBFA2T3- GLIS2 fusion gene in pediatric AML.
In the present study, we investigated the in vitro effects of GANT61 on AML cell lines and primary cells from AML patients harboring the CBFA2T3- GLIS2 fusion gene.
The materials and methods are detailed in Additional file 1. Molecular analysis of CBFA2T3- GLIS2 fusion gene is reported in Additional file 2: Figure S1. Genetic features of control AML cell lines without GLIS2 fusion are reported in Additional file 3: Table S1.
Our results showed that AML cell lines with CBFA2T3- GLIS2 fusion gene have a higher sensitivity to GANT61 than other AML cell lines without this genetic aberration (Fig.  1a). Similar results were obtained on primary leukemia cells isolated from AML patients, being the IC 50 of the GLIS2-positive leukemia and negative primary cells 13.6 and 41.6 μM, respectively (Fig.  1b).
Treatment with GANT61 induced an increase of about 30% of apoptotic cells (Fig.  1c) and block of cell cycle in G0/G1 phase only in M07e and WSU-AML lines positive to CBFA2T3- GLIS2 (Fig.  1d and Additional file 4: Figure S2).
We further analyzed the expression profile of cell lines and primary cells following GANT61 treatment. Through qPCR, we demonstrated that GANT61 treatment led to a significant reduction of the expression of BMP2 and GLIS2 (Fig.  2a, b). In order to fully characterize the effect of GANT61 treatment on whole transcriptome profile of CBFA2T3- GLIS2-positive cells, gene expression was assessed by microarray analysis. After GANT61 treatment, the expression of target genes of CBFA2T3- GLIS2, such as CRISP3, GATA3, H2AFY, or NCAM1(CD56) was significantly downregulated ( p < 0.05) (Fig.  2c). Moreover additional genes were downregulated by the treatment with GANT61 ( p < 0.05), as for example, genes involved in cell cycle control, such as CDC25A, CDC7, CDCA2, and CCNA2. The expression of these genes is required for progression through cell cycle, and their expression is aberrant in AML, as well as in other malignancies [ 9, 10]. In our model, the expression of these genes is downregulated following GANT61 treatment, this partly explaining the cell cycle arrest observed in AML cell lines with GLIS2 fusion after GANT61 treatment (Fig.  1d). Genes involved in cell proliferation ( KIF14, MELK, MCM10, NUF2) and epigenetic regulators namely DNMT1 and DNMT3B were also present. Considering the particular interest of these DNA methyltransferase genes, we performed chromatin immunoprecipitation (ChIP) analysis using a CBFA2T3-specific antibody on WSU-AML and M07e cell lines. Our findings showed that CBFA2T3-GLIS2 fusion protein directly binds to the proximal promoter of DNMT1 and DNMT3B, positively regulating their expression (Fig.  2d). Overexpression of DNMT genes could lead to DNA hypermethylation and could be involved in the leukemogenesis process.
Since in the classic Hedgehog signaling pathway, several target genes involved in feedback mechanisms ( HHIP, PTCH1, GLI1) have been described; we hypothesized that CBFA2T3-GLIS2 chimeric protein could regulate wild-type GLIS2 protein with the same feedback mechanism. Western blotting analysis confirmed the presence of chimeric protein and showed also the presence of wild-type GLIS2. These results may point to the role of the fusion protein in regulating wild-type GLIS2. Following GANT61 treatment, the expression of both proteins was decreased with respect to untreated samples, suggesting that GANT61 treatment targeted both CBFA2T3-GLIS2 fusion protein and also wild-type GLIS2 expression (Fig.  2e). Moreover, other AML cell lines not carrying the CBFA2T3- GLIS2 fusion gene did not show expression of GLIS2 (data not shown).
On the other hand, western blotting analysis showed that expression of GLI1 and GLI2 did not decrease following GANT61 treatment (Additional file 5: Figure S3).
Another study demonstrated a high sensitivity of this subgroup of AML with GLIS2 fusion to Aurora A kinase (AURKA) inhibitors [ 4]. We therefore investigated the effect of GANT61 and AURKA inhibitor MK-0457 in M07e and WSU-AML cell lines carrying the CBFA2T3- GLIS2 fusion gene. Cell lines were incubated for 48 h with either single drugs or a combination of the two drugs at a constant ratio of 1:10 (GANT61:MK-0457). The combined treatment showed a higher cytotoxic effect when compared to each single drug, and the two inhibitors displayed a synergistic effect on cell growth, as indicated by the CI value (Additional file 6: Figure S4).
This work provides preliminary data from preclinical in vitro and ex vivo studies targeting pediatric AML with CBFA2T3- GLIS2 fusion gene. Although further investigation will be required to confirm these results, our experience with GANT61 represents a preliminary background for further evaluating in vivo the inhibition of GLIS2-mediated transcription in AML harboring the CBFA2T3- GLIS2 fusion gene.

Acknowledgements

The authors would like to thank Dr. Tanja Gruber from St. Jude Children’s Research Hospital, Memphis, for kindly providing the WSU-AML cell line.

Funding

This work was partly supported by the grants from Fondazione Veronesi (Young Investigator Grant, to R. Masetti), AIRC (Associazione Italiana Ricerca sul Cancro, My First AIRC grant to R. Masetti), AIRC (Associazione Italiana Ricerca sul Cancro, Special Grant “5xmille”-9962 to F. Locatelli), Ministero della Salute (RF-2010-2316606 to F. Locatelli; Ricerca Corrente to F. Locatelli) and Ministero dell’Istruzione, Università e Ricerca (Grant Progetto di Rilevante Interesse Nazionale, PRIN 2010, to F. Locatelli), Cariparo-Fondazione città della Speranza to M. Pigazzi and G. Basso, and PRAT-Università degli Studi di Padova to M. Pigazzi.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

MR and SNB designed the work, performed the data analysis, and wrote the manuscript; AA contributed to write the manuscript; SNB, AA, FC, AL, JB, SS, MF, and MP performed the laboratory work for this study; MD and VI performed the statistical and bioinformatics analyses; AMM, GB, FL, and AP supervised the work and contributed to the critical revision; AP, designed and supervised the research. All authors read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The samples analyzed have been obtained from patients enrolled in the Associazione Italiana Ematologia Oncologia Pediatrica 2002/01 clinical trial, which was approved by the institutional review board of the Sant’Orsola-Malpighi Hospital, Bologna, Italy. The patients gave informed consent in accordance with the Declaration of Helsinki.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://​creativecommons.​org/​licenses/​by/​4.​0/​), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.

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