Characterization Of A Novel Protein-Coding Gene Named Tihl (Translocated In Hodgkin'S Lymphoma)

Previously, we described the CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011) was highly expressed in Hodgkin9s Lymphoma cell line KM-H2. The Class II Transactivator (CIITA) has been described to function in the regulation of immune responses and its deregulation may serve as a mechanism through which tumor cells evade immunosurveillance. In contrast, the expression and function of the gene/protein encoded by the novel locus BX648577/FLJ27352/C15orf65 is unknown. In the present study, we report the endogenous expression and describe the biological function of the novel gene which we named TIHL (Translocated in Hodgkin9s Lymphoma). Comparative analysis in various species revealed high evolutionary conservation (≥50%) between human, mouse, gorilla and Drosophila TIHL orthologs. Using quantitative real time-PCR, we detected expression of full-length TIHL in both healthy and malignant hematopoietic cells. By immunoblot, we detected robust expression of the 13-16 kDa protein in leukemia and lymphoma cell lines using a TIHL-specific antibody. Lentiviral expression of TIHL in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells led to enhanced clonogenicity and increased proliferative capacity with significant increases in the integration of EdU (5-ethynyl-2′-deoxyuridine) and the percentage of cells in S-phase. Likewise, TIHL knockdown using 2 independent lentiviral shRNAs, led to decreased proliferation of both KG1a and NB4 leukemia cells grown in either suspension culture or semi-solid media. Ingenuity Pathway Analysis (IPA) after microarray analysis of NB4 cells overexpressing TIHL highlights significantly altered functional categories including ‘cell growth and proliferation,’ ‘cell death and survival,9 and ‘cell cycle,9 in line with our previous observations. In silico studies, predicted transcriptional regulators of TIHL including the gene GLI1 (Glioma-Associated Oncogene Homolog 1). GLI1, a positive regulator of the Hedgehog signaling pathway, targets genes involved in cell cycle regulation, cell proliferation and cell death. Treatment of KG1a and NB4 leukemia cells with the small molecule GLI inhibitor GANT61 led to significant inhibition of cell proliferation. Additionally, TIHL mRNA expression was significantly downregulated at increasing concentrations of GANT61 by qRT-PCR. Preliminary data suggests that TIHL overexpression may partially rescue the leukemia inhibitory effects of GANT61 treatment. These data strongly suggest that the novel gene TIHL is involved in cell cycle regulation/modulation in hematopoietic cells. Further studies will involve analysis of the effects of TIHL overexpression or knockdown in healthy donor-derived bone marrow cells, chromatin immunoprecipitation (ChIP)-PCR to confirm its direct transcriptional regulation by GLI1 and co-IP coupled to mass spectrometry to identify direct protein partners of this novel gene. Citation Format: Ujunwa C. Okoye-Okafor, Laura Barreyro, Mario Pujato, Heng Rui Wang, Boris Bartholdy, Britta Will, Tihomira I. Todorova, Masahiro Kawahara, Bruce Woolcock, Andras Fiser, Randy Gascoyne, Christian Steidl, Ulrich Steidl. Characterization of a novel protein-coding gene named TIHL (Translocated in Hodgkin9s Lymphoma). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2014-2945