gant-61 and Leukemia--Myeloid--Acute

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma-associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinases; Cytarabine; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Leukemic; Glycogen Synthase Kinase 3; HEK293 Cells; Humans; Leukemia, Myeloid, Acute; Mice, Nude; Phosphatidylinositol 3-Kinase; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Signal Transduction; THP-1 Cells; Tumor Burden; U937 Cells; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

Myelodysplastic syndromes (MDSs) are stem cell disorders with risk of transformation to acute myeloid leukemia (AML). Gene expression profiling reveals transcriptional expression of GLI1, of Hedgehog (Hh) signaling, in poor-risk MDS/AML. Using a murine model of MDS we demonstrated that constitutive Hh/Gli1 activation accelerated leukemic transformation and decreased overall survival. Hh/Gli1 activation resulted in clonal expansion of phenotypically defined granulocyte macrophage progenitors (GMPs) and acquisition of self-renewal potential in a non-self-renewing progenitor compartment. Transcriptome analysis of GMPs revealed enrichment in gene signatures of self-renewal pathways, operating via direct Gli1 activation. Using human cell lines we demonstrated that in addition to canonical Hh signaling, GLI1 is activated in a Smoothened-independent manner. GLI1 knockdown or inhibition with GANT61 resulted in decreased proliferation and clonogenic potential. Our data suggest that GLI1 activation is frequent in MDS during disease progression and inhibition of GLI1 is an attractive therapeutic target for a subset of patients.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Granulocyte-Macrophage Progenitor Cells; Hedgehog Proteins; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Pyridines; Pyrimidines; Zinc Finger Protein GLI1

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.

Topics: Apoptosis; Cell Cycle Checkpoints; Child; Down-Regulation; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Leukemia, Myeloid, Acute; Oncogene Proteins, Fusion; Pyridines; Pyrimidines; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Proteins; Zinc Finger Protein GLI1

Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61.. The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61.. Kasumi-1 and KG1a cells, containing more CD34+ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34+ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34+ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34+ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity.. The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

Topics: Adolescent; Adult; Aged; Antigens, CD34; Apoptosis; Bone Marrow Cells; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; G1 Phase Cell Cycle Checkpoints; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplastic Stem Cells; Pyridines; Pyrimidines; RNA Interference; RNA, Small Interfering; Young Adult; Zinc Finger Protein GLI1

The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML).. Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo.. Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice.. GLI expression is a negative prognostic factor and might represent a novel druggable target in AML.

Topics: Adolescent; Adult; Animals; Biomarkers, Tumor; Case-Control Studies; Cell Line, Tumor; Disease-Free Survival; DNA Mutational Analysis; Female; Hedgehog Proteins; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Mice, Inbred NOD; Mice, SCID; Middle Aged; Neoplasm Transplantation; Prognosis; Proportional Hazards Models; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Transcription Factors; Treatment Outcome; Young Adult; Zinc Finger Protein GLI1