Tyrosine Kinase Non Receptor 2 Research Articles

ZNF692 promotes osteosarcoma cell proliferation, migration, and invasion through TNK2-mediated activation of the MEK/ERK pathway

BackgroundOsteosarcoma is a diverse and aggressive bone tumor. Driver genes regulating osteosarcoma initiation and progression remains incompletely defined. Zinc finger protein 692 (ZNF692), a kind of Krüppel C2H2 zinc finger transcription factor, exhibited abnormal expression in different types of malignancies and showed a correlation with the clinical prognosis of patients as well as the aggressive characteristics of cancer cells. Nevertheless, its specific role in osteosarcoma is still not well understood.MethodsWe investigated the dysregulation and clinical significance of ZNF692 in osteosarcoma through bioinformatic method and experimental validation. A range of in vitro assays, including CCK-8, colony formation, EdU incorporation, wound healing, and transwell invasion tests, were conducted to assess the impact of ZNF692 on cell proliferation, migration, and invasion in osteosarcoma. A xenograft mouse model was established to evaluate the effect of ZNF692 on tumor growth in vivo. Western blot assay was used to measure the protein levels of MEK1/2, P-MEK1/2, ERK1/2, and P-ERK1/2 in cells that had been genetically modified to either reduce or increase the expression of ZNF692. The relationship between ZNF692 and tyrosine kinase non-receptor 2 (TNK2) were validated by qRT-PCR, chromatin immunoprecipitation and luciferase reporter assays.ResultsExpression of ZNF692 was increased in both human osteosarcoma tissues and cell lines. Furthermore, the expression of ZNF692 served as an independent predictive biomarker in osteosarcoma. The results of the survival analysis indicated that increased expression of ZNF692 was associated with worse outcome. Downregulation of ZNF692 inhibits the proliferation, migration, and invasion of osteosarcoma cells, whereas upregulation of ZNF692 has the opposite impact. Western blot assay indicates that reducing ZNF692 decreases phosphorylation of MEK1/2 and ERK1/2, whereas increasing ZNF692 expression enhances their phosphorylation. U0126, a potent inhibitor specifically targeting the MEK/ERK signaling pathway, partially counteracts the impact of ZNF692 overexpression on the proliferation, migration, and invasion of osteosarcoma cells. In addition, ZNF692 specifically interacts with the promoter region of TNK2 and stimulates the transcription of TNK2 in osteosarcoma cells. Forcing the expression of TNK2 weakens the inhibitory impact of ZNF692 knockdown on P-MEK1/2 and P-ERK1/2. Similarly, partly inhibiting TNK2 counteracts the enhancing impact of ZNF692 overexpression on the phosphorylation of MEK1/2 and ERK1/2. Functional tests demonstrate that the suppressive effects of ZNF692 knockdown on cell proliferation, migration, and invasion are greatly reduced when TNK2 is overexpressed. In contrast, the reduction of TNK2 hinders the ability of ZNF692 overexpression to enhance cell proliferation, migration, and invasion.ConclusionZNF692 promotes the proliferation, migration, and invasion of osteosarcoma cells via the TNK2-dependent stimulation of the MEK/ERK signaling pathway. The ZNF692-TNK2 axis might potentially function as a possible predictive biomarker and a promising target for novel therapeutics in osteosarcoma.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Histoepigenetic analysis of the mesothelin network within pancreatic ductal adenocarcinoma cells reveals regulation of retinoic acid receptor gamma and AKT by mesothelin

To enable computational analysis of regulatory networks within the cancer cell in its natural tumor microenvironment, we develop a two-stage histoepigenetic analysis method. The first stage involves iterative computational deconvolution to estimate sample-specific cancer-cell intrinsic expression of a gene of interest. The second stage places the gene within a network module. We validate the method in simulation experiments, show improved performance relative to differential expression analysis from bulk samples, and apply it to illuminate the role of the mesothelin (MSLN) network in pancreatic ductal adenocarcinoma (PDAC). The network analysis and subsequent experimental validation in a panel of PDAC cell lines suggests AKT activation by MSLN through two known activators, retinoic acid receptor gamma (RARG) and tyrosine kinase non receptor 2 (TNK2). Taken together, these results demonstrate the potential of histoepigenetic analysis to reveal cancer-cell specific molecular interactions directly from patient tumor profiles.

Identification and Characterization of Tyrosine Kinase Nonreceptor 2 Mutations in Leukemia through Integration of Kinase Inhibitor Screening and Genomic Analysis.

The amount of genomic information about leukemia cells currently far exceeds our overall understanding of the precise genetic events that ultimately drive disease development and progression. Effective implementation of personalized medicine will require tools to distinguish actionable genetic alterations within the complex genetic landscape of leukemia. In this study, we performed kinase inhibitor screens to predict functional gene targets in primary specimens from patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Deep sequencing of the same patient specimens identified genetic alterations that were then integrated with the functionally important targets using the HitWalker algorithm to prioritize the mutant genes that most likely explain the observed drug sensitivity patterns. Through this process, we identified tyrosine kinase nonreceptor 2 (TNK2) point mutations that exhibited oncogenic capacity. Importantly, the integration of functional and genomic data using HitWalker allowed for prioritization of rare oncogenic mutations that may have been missed through genomic analysis alone. These mutations were sensitive to the multikinase inhibitor dasatinib, which antagonizes TNK2 kinase activity, as well as novel TNK2 inhibitors, XMD8-87 and XMD16-5, with greater target specificity. We also identified activating truncation mutations in other tumor types that were sensitive to XMD8-87 and XMD16-5, exemplifying the potential utility of these compounds across tumor types dependent on TNK2. Collectively, our findings highlight a more sensitive approach for identifying actionable genomic lesions that may be infrequently mutated or overlooked and provide a new method for the prioritization of candidate genetic mutations.

Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancer uncovers rare somatic mutations in TNK2 and DDR1.

BackgroundEndometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets.MethodsIn a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines.ResultsOverall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2P761Rfs*72 frameshift mutation was recurrent in EC, and the DDR1R570Q missense mutation was recurrent across tumor types.ConclusionsThis is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-884) contains supplementary material, which is available to authorized users.

Integrated analysis of RNAi screens in pediatric rhabdomyosarcoma.

10040 Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Embryonal RMS (ERMS) are characterized by 11p15 LOH, while alveolar RMS (ARMS) harbor a translocation between PAX3 or PAX7 and FOXO1. Relapsed or metastatic RMS has a poor prognosis with a 5-year survival rate of 25%. We hypothesized that a reduction in proliferation of ARMS cell lines as result of the loss of function (LOF) of a specific protein via RNAi likely point to novel targets that exhibit “synthetic lethality” with the PAX3/7-FOXO1 translocation, and can be used to reveal unique ARMS vulnerabilities. Methods: We are using two complementary screening strategies to identify genes critical for RMS cell growth and survival. One RNAi screening strategy used an inducible LOF shRNA screen of 15,000 shRNA -5,000 genes- introduced into RH30 (ARMS) or RD (ERMS) cells. Data analysis after subtraction of targets related to common survival pathways revealed 40 genes with close association with RMS cell growth. Follow up analysis included further comparison of the effects of gene specific LOF in ARMS and ERMS cell lines and assessment in orthotopic xenografts. To increase the strength of our findings, we have also conducted parallel siRNA screens of the human kinome targeting 704 kinase genes (3 siRNAs per gene) in RH30 or RD cells. Results: Of the 40 genes identified by shRNA screening, 15 genes were identified as selectively needed for ARMS cell growth. Follow up analysis of one of these genes, TNK-2 (tyrosine kinase, non-receptor 2) in orthotopic xenografts, confirmed that LOF of TNK2 markedly decreases ARMS cell growth. The siRNA screen has identified a further putative 26 genes that selectively reduce ARMS cell growth. These screens have led us to focus on members of the ERK, PI3K and IGF1R pathways as potential targets for the treatment of ARMS. Conclusions: RNAi screening affords an unbiased method for the discovery and elucidation of gene function. Ongoing efforts are focused on the integration of results obtained to gain further insight into the signaling networks influencing alveolar rhabdomyosarcoma biology and thus identify a small group of druggable genes that could represent alveolar rhabdomyosarcoma specific vulnerabilities.

The activation mechanism of ACK1 (activated Cdc42-associated tyrosine kinase 1)

ACK [activated Cdc42 (cell division cycle 42)-associated tyrosine kinase; also called TNK2 (tyrosine kinase, non-receptor, 2)] is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein-coupled receptor signalling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In the present study, we demonstrated that interaction of the SH3 (Src homology 3) domain with the EBD [EGFR (epidermal growth factor receptor)-binding domain] in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1, independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (Leu10-Leu14) is essential for cell adhesion-mediated activation. In the activation of ACK1 by EGFR signalling, Grb2 (growth-factor-receptor-bound protein 2) mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of Ser445 to proline caused constitutive activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.

Identification of TNK2 as a critical kinase in rhabdomyosarcoma through a loss of function shRNA screen.

9511 Background: Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma. Embryonal rhabdomyosarcomas (ERMS) are characterized by 11p15 LOH while alveolar rhabdomyosarcomas harbor a translocation between PAX3 or PAX7 and FOXO1. Relapsed or metastatic disease has a 5-year survival rate of 25%. Methods: We sought to identify critical genes for rhabdomyosarcoma cell growth and survival. We performed a loss-of-function shRNA screen where a library of 15,000 shRNAs was introduced in RH30 (ARMS) and RD (ERMS) cells engineered to express the bacterial tetracycline repressor in a tet-on system. Relative abundance of cells containing a certain shRNA was determined using specific barcodes. We subtracted “hits” that are common to 4 lymphoma cell-lines to eliminate common survival pathways. Forty genes were associated with rhabdomyosaroma cell growth, of which 15 cause greater suppression in ARMS. We performed a subsequent validation of these 15 genes in multiple ARMS and ERMS cell lines using three different shRNA sequences using lentiviral constructs. In addition, we validated our results in an independent siRNA screen of the protein kinome in RMS cells. Results: TNK2 (tyrosine kinase, non-receptor 2) was identified as tyrosine kinase involved in rhabdomyosarcoma cell growth independently in the inducible shRNA screen and in the non-inducible siRNA platform. This molecule has been involved in downstream signaling from EGFR and integrins. Amplification of the TNK2 gene, located at 3q29, has been described in breast, prostate and lung cancer. More recently, a siRNA screen identified it as a potential therapeutic target in Ewing’s sarcoma. We confirmed TNK2 expression across patient tumor samples in a publically available database; where patients with high TNK2 expression showed lower overall survival. TNK2 knock down resulted in decreased rhabdomyosarcoma cell growth in vitro and in orthotopic xenografts. We confirmed specificity of our findings with a rescue experiment. Conclusions: We identified TNK2 as a potential therapeutic target in rhabdomyosarcoma using a loss-of-function shRNA screen and subsequent validation. Further work is ongoing to fully characterize the molecular mechanisms involved in these findings.