Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene
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CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins.
We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile.
As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions.
Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.
KeywordsAcute myeloid leukemia Acute megakaryoblastic leukemia CBFA2T3-GLIS2 GANT61 Hedgehog pathway
Acute myeloid leukemia
Aurora A kinase
Bone morphogenic protein
GLI-similar protein 2
4-[3-(4 lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate)
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 [1, 2, 3]. The incidence of this aberration is 17 and 8% in pediatric non-Down syndrome acute megakaryoblastic and in pediatric cytogenetically normal AML, respectively [1, 2, 3]. 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 [5, 6, 7, 8]. 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.
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).
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 . 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.
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.
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.
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.
The authors declare that they have no competing interests.
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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.
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