The GLI-Code As a Potential Therapeutic Target in Myeloid Neoplasms

Abstract

The proliferation of hematopoietic stem cells (HSCs) and progenitor cells (HPC) is tightly regulated during physiologic function of the hematopoietic system. Recent data suggest a role for activated developmental signaling pathways such as Hedgehog (Hh), Notch and Wnt in this process. The GLI genes (GLI1-4), known as GLI-code, encode Cys2-His2 zinc finger transcription factors that regulate the expression of target genes at the distal end of the canonical Hh pathway. This pathway is important during embryogenesis but mostly inactive in normal adult tissues. Activation of Hh effector SMO triggers an intracellular signaling cascade leading to activation of GLI2 and GLI3, which ultimately activate GLI1. It is well established that activation of GLI transcription factors may occur independent of canonical Hh signaling. Aberrant activation of the Hh/GLI pathway has been implicated in tumor progression and resistance to chemotherapy in MDS/AML. GLI transcription factors serve as a common node of activation through which many oncogenic signals, e.g., Hh, FLT3, PI3K-AKT, TGFβ and RAS converge. Moreover, overexpression of GLI transcription factors constitutes a negative prognostic survival marker. Higher expression of GLI transcription factors were recently reported in mutant FLT3-ITD AML cases. Extensive analysis of publicly available gene expression data sets revealed that GLI1 expression was significantly higher in complex AML karyotype (p<0.001, n=48) compared to other cytogenetic abnormalities t(15,17), inv(16), t(8,21), and t(11q23) (n=54, 47, 60 and 43 respectively). GLI1 expression was also significantly higher (p<0.05) in complex AML karyotype (n=87) compared to normal karyotype cases (n=989). The expression of GLI1 was even higher (p<0.001) in MDS (n=228) than normal AML karyotype. In addition, 8 out of 14 of NRAS mutant and 5 out of 7 KRAS mutant cases of AML in TCGA database have higher GLI1 expression. These biomarkers indicate advanced disease and constitute a major clinical problem with limited therapeutic options. Our observations strongly suggest that the GLI1 would be a bona-fide therapeutic target in MDS/AML. Thus specific and potent inhibitors of GLI1 can provide a rational approach for the treatments of these cases.While most efforts have been directed towards therapeutic targeting of upstream Hh signaling through PTCH/SMO, these measures remain ineffective in the presence of parallel signaling converging at GLI independent of canonical Hh pathway. Therefore, there is a need for the development of a GLI1-specific inhibitor that effectively blocks the nodal channel of Hh signaling. For this purpose, we used a structure guided approach to develop a highly potent and specific GLl1/GLI2 inhibitor iGli702, capable of preventing GLI1 DNA binding and thereby induction of cell death. Computational, biochemical and biophysical analysis demonstrated that iGli702 binds to the DNA binding surface of GLI1 protein between Zn-finger 2 and 3, effectively preventing Gli1-DNA interaction. Our data show that iGli702 is over 40 fold more effective than the reported GLI1/2 antagonist GANT61 in AML cell lines. iGli702 promotes apoptosis in a panel of leukemic cell lines, primary patient-derived AML samples and animal models. We observed an IC50 of 150 nM in cell viability assays.To test whether iGli702 induces cell death of progenitor tumor cells in semi-solid methylcellulose-based media, sorted CD34 positive tumor cells from AML cell lines (MOLM-13 and THP1) were treated with iGli702. This treatment significantly reduced colony formation of leukemia cells. However, iGli702 has only a minimal effect on normal PBMC and BM cells (LD50 >10µM). iGli702 efficacy was also compared in vitro to common chemotheraputic agents in AML. CytA and Ida were applied in parallel with iGli702to MOLM-13, THP-1, K562 AML cell lines. iGli702 showed superior and equivalent activity to CytA and Ida, respectively. In addition, we applied deep RNA NGS to analyze the cellular transcriptome after treatment with iGli702. Our data suggest that iGli702 directly inhibits Hedgehog pathway by reducing Hh target genes expression. In summary, iGli702 is highly effective as a single agent in killing hematopoietic malignant cells. This in turn demonstrates that targeting GLI1 Hh effector node is an excellent therapeutic approach in myeloid neoplasms. DisclosuresNo relevant conflicts of interest to declare.