Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia

Abstract

Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.

Understanding the Hedgehog Signaling Pathway in Acute Myeloid Leukemia Stem Cells: A Necessary Step toward a Cure

“…One way to target GLI proteins could be by the activation of GLI3 R using hypomethylating agents [ 80 ]. Additionally, low levels of GLI3 result in a protective effect from the cytotoxic effects of Ara-C on LSCs or progenitors cells [ 82 ].…”

“…Moreover, they found that there was no expression of HH ligands in their cohort [ 81 ]. Recently, the group suggested that the downregulation of GLI3 may be associated with a low sensitivity to cytarabine (Ara-C) [ 82 ], which may indicate that low levels of this protein protect LSCs or progenitor cells from the effect of Ara-C. Furthermore, other studies that targeted the refractory/relapse population in AML have seen that overexpression of GLI1 leads to an increase in drug resistance [ 83 ].…”

Mebendazole Mediates Proteasomal Degradation of GLI Transcription Factors in Acute Myeloid Leukemia

“…Protein isolation and western blots were performed as described before [ 48 ]. For analysis of phosphorylated proteins, cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (89900, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (cOmpleteTM Tablets, 11697498001, Roche, Basel, Swiss; Sodium orthovanadate, 13721-39-6, Fivephoton Biochemicals, San Diego, CA, USA).…”

Inhibitors of Chemoresistance Pathways in Combination with Ara-C to Overcome Multidrug Resistance in AML. A Mini Review

“…ABCC4 inhibition can be accomplished with the use of inhibitors such as sorafenib or MK571 or by silencing it with siRNA ( Figure 3 b). Unfortunately, this strategy also sensitizes parental myeloids to Ara-C; therefore, it also induces toxicity and exacerbates hematological damage in healthy cells [ 51 , 79 , 80 , 81 , 82 , 83 ]. This is a serious drawback and remains a challenge to be addressed.…”