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

Abstract

Background 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.MethodsWe 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.ResultsAs 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.ConclusionsOur findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0396-0) contains supplementary material, which is available to authorized users.

CBFA2T3‐GLIS2‐positive acute myeloid leukaemia. A peculiar paediatric entity

“…Using ChIP, our group showed a significant enrichment of CBFA2T3‐GLIS2 fusion protein on the NCAM1 ( CD56 ) promotor, confirming that CBFA2T3‐GLIS2 directly regulates the expression of CD56 (Masetti et al , ).…”

“…Treatment with GLI inhibitors interferes with expression pathway induced by CBFA 2T3‐ GLIS 2. The specific interaction between GANT 61 and GLIS 2 remains to be formally demonstrated (Masetti et al , ). Di MF , dimethylfasudil.…”

“…These data support the concept that the NHR2 domain is essential for the establishment of CBFA2T3‐GLIS2 transcriptional alterations and for the in‐vivo maintenance of human CBFA2T3‐GLIS2 ‐expressing AMKL cells. In recent years, our group has been working to specifically counteract the leukaemia‐promoting effect of the fusion gene by inhibiting its binding to DNA through targeting of the GLIS2 zinc finger domain (Masetti et al , ). Indeed, the GLIS2 protein shares a highly homologous zinc finger domain with members of the GLI proteins (Vasanth et al , ).…”

“…The GLI inhibitor GANT61 is a small molecule that inhibits DNA binding of GLI family proteins, and it is mainly used in preclinical studies (Pan et al , ; Wellbrock et al , ). 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 in childhood AML (Masetti et al , ). Experimental results showed that, after exposure to GANT61, both cell lines and primary AML cells carrying CBFA2T3 ‐ GLIS2 showed a higher sensitivity to undergo apoptosis and to display G1 cell‐cycle arrest than AML cells without the GLIS2 fusion (Masetti et al , ) (Fig ).…”

“…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 in childhood AML (Masetti et al , ). Experimental results showed that, after exposure to GANT61, both cell lines and primary AML cells carrying CBFA2T3 ‐ GLIS2 showed a higher sensitivity to undergo apoptosis and to display G1 cell‐cycle arrest than AML cells without the GLIS2 fusion (Masetti et al , ) (Fig ). Moreover, GANT61 treatment induced down‐regulation of some genes directly regulated by the fusion gene, including ERG , GATA3 , DNMT1 and DNMT3B , suggesting the specificity of GANT61 treatment to block the DNA binding (Masetti et al , ).…”