A novel mechanism of regulation of the transcription factor GLI3 by toll-like receptor signaling

The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal exclusively through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using IRF3−/− MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3−/−), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF-α compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

GLI3 Is Associated With Neuronal Differentiation in SHH-Activated and WNT-Activated Medulloblastoma

Glioma-associated oncogene homolog 3 (GLI3), whose main function is to inhibit GLI1, has been associated with neuronal differentiation in medulloblastoma. However, it is not clear what molecular subtype(s) show increased GLI3 expression. GLI3 levels were assessed by immunohistochemistry in 2 independent cohorts, including a total of 88 cases, and found to be high in both WNT- and SHH-activated medulloblastoma. Analysis of bulk mRNA expression data and single cell RNA sequencing studies confirmed that GLI1 and GLI3 are highly expressed in SHH-activated medulloblastoma, whereas GLI3 but not GLI1 is highly expressed in WNT-activated medulloblastoma. Immunohistochemical analysis has shown that GLI3 is expressed inside the neuronal differentiated nodules of SHH-activated medulloblastoma, whereas GLI1/2 are expressed in desmoplastic areas. In contrast, GLI3 is diffusely expressed in WNT-activated medulloblastoma, whereas GLI1 is suppressed. Our data suggest that GLI3 may be a master regulator of neuronal differentiation and morphology in these subgroups.

Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia

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.

GLI3 repressor determines Hedgehog pathway activation and is required for response to SMO antagonist glasdegib in AML

Key Points GLI3R inhibits Hh signaling and is required for response to SMO antagonist in AML. GLI3 is silenced in AML, and decitabine restores GLI3 expression and leads to modulation of Hh signaling.