Constitutively Activated STAT3 Induces the Expression of Gli1 in Chronic Lymphocytic Leukemia Cells
Authors: Rozovski Uri , Harris David M. , Li Ping , Liu Zhiming , Manshouri Taghi , Veletic Ivo , Jain Preetesh , Ferrajoli Alessandra , Burger Jan A. , Bose Prithviraj , Thompson Philip A. , Jain Nitin , Verstovsek Srdan , Wierda William G. , Keating Michael J , Estrov Zeev E. 
Published: November 2019

The transcription factor glioma associated oncogene (Gli)-1 is a downstream effector of the Hedgehog (HH) signaling pathway. The HH pathway plays a critical role in embryonic development, and in maintenance of stem cells, tissue homeostasis, hematopoiesis, and immune response in the adult organism. Constitutive activation of the HH pathway has been implicated in the development of various cancer types. A recent study of blood samples from 841 treatment-naïve CLL patients identified activated Gli1 in 49%, and mutations in HH pathway genes, that induce activation of Gli1, in 11% of the patients (Ghia et al. Blood 1019). However what induces the expression of Gli1 in CLL cells that do not carry a mutation(s) in the HH pathway has not been elucidated.
Because inhibition of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-3 pathway in liver cells inhibits the transcription of Gli1 (Zhang et al. J Cell Mol Med 2018), and because in CLL cells STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3, we postulated that constitutively activated STAT3 induces the expression of Gli1 in CLL cells.
To test our hypothesis we first performed western immunoblotting and found high levels of Gli1 in cell lysates from 6 of 7 CLL patients but not in normal B cells, and using flow cytometry we found that 76% of CD5+/CD19+ CLL cells expressed intracellular Gli1 and that intracellular phosphoserine STAT3 and Gli1 are co-expressed in 70.4% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of Gli1 and of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Using sequence analysis we identified in the Gli1 gene promoter 5 γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites, spanning within 930 base pairs upstream the Gli1 start codon. To determine which putative binding site binds STAT3 we designed 5 different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated 4 of 5 STAT3-putative binding sites at high affinity. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-Gli1 DNA complexes with DNA probes designed to detect the PTX3 gene promoter STAT3-binding sites and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the Gli1 gene promoter. To further confirm these findings, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and Gli1 mRNA levels.
Because Gli1 expression was associated with disease progression (Ghia et al. Blood 1019), we wondered whether Gli1 provides CLL cells with survival advantage and whether inhibition of Gli1 would affect the viability of CLL cells. To answer this question we transfected CLL cells from two different treatment-naïve CLL patients with Gli1 small interfering (si)RNA and, using annexin V/PI, found that Gli1-siRNA induced apoptosis of CLL cells. To further confirm these data we incubated CLL cells with GANT61, a small molecule that inhibits Gli1, and, as previously reported, found that GANT61 increases the spontaneous apoptosis rate of CLL cells, suggesting that inhibition of Gli1 might benefit patients with CLL.
In conclusion, we found that constitutively phosphorylated STAT3 activates the Gli1 gene promoter and induced the production of Gli1 protein in CLL cells. In addition, we found that downregulation of Gli1 expression or inhibition of Gli1 induced apoptosis of CLL cells, suggesting that Gli1 is a target for therapy of CLL.

Disclosures
Burger: BeiGene: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Gilead Sciences: Research Funding. Bose:Blueprint Medicine Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Incyte: Research Funding; Roche: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; Promedior: Research Funding. Wierda:Gilead Sciences: Research Funding; Cyclcel: Research Funding; Sunesis: Research Funding; Acerta Pharma Inc: Research Funding; AbbVie: Research Funding; KITE pharma: Research Funding; Xencor: Research Funding; Janssen: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics LLC: Research Funding; Loxo Oncology Inc.: Research Funding; Oncternal Therapeutics Inc.: Research Funding.