Identification Of Kinase Targets Research Articles

Abstract 80: Utilizing a decellularized PDX tumor matrix model to investigate LKB1 regulation of ECM remodeling in TNBC

Abstract Triple negative breast cancer (TNBC) is an aggressive disease with poor prognoses, partly due to the lack of clinically approved targeted therapeutics. The mechanisms underlying the process of drug resistance in TNBC remain elusive, prompting the need for the identification of kinase targets. Our lab previously found that liver kinase B1 (LKB1) overexpression inhibits the migratory phenotype and pathways involved in EMT of TNBC. However, the specific mechanisms in which LKB1 suppresses TNBC metastasis have not been fully elucidated. We believe that overexpression of LKB1 inhibits the migratory axis by inhibiting ECM remodeling that promotes invasion in TNBC. Here we utilize our novel decellularized patient-derived xenograft (PDX) TNBC model, TU-BcX-2K1 (“2K1”) tumor scaffold, to test this hypothesis. We seeded MDA-MB-231-LKB1 overexpressed and MDA-MB-231-vector cells onto decellularized 2K1 PDX tumor scaffold or decellularized adipose tissue (“AT”) and implanted our model into SCID/beige mice. Results demonstrated that tumor matrix enhanced cancer cell growth in both MDA-MB-231-vector and -LKB1 cell lines. LKB1 on adipose scaffold did not have tumor formation. These data suggest a novel microenvironment dependent function of LKB1. We compared the number of metastases in the lungs and livers excised from mice in each group. There were significantly more lung metastases in the 2K1+MDA-MB-231-vector group than in the AT+MDA-MB-231-vector group (p-value = 0.0144). There were slightly more lung metastases in the AT-MDA-MB-231-LKB1 group than in the 2K1+MDA-MB-231-LKB1 group, albeit not significantly. There were more liver metastases in the 2K1+MDA-MB-231-vector group than in the AT+MDA-MB-231-vector group, albeit not significantly. There were also more liver metastases in the 2K1+MDA-MB-231-LKB1 group than in the AT+MDA-MB-231-LKB1 group; however, there was no significance. LKB1 appeared to inhibit stromal remodeling in tumor, evident from decreased collagen composition and stromal cell infiltration. In vitro validation of this demonstrated that LKB1 secretome was able to inhibit adipogenesis of stem cells. To further test LKB1's role in stromal remodeling, we performed an in vivo experiment co-injecting MDA-MB-231-LKB1 and MDA-MB-231-vector cells with ASCs into SCID/beige mice. MDA-MB-231-LKB1 tumors grew significantly slower than MDA-MB-231-vector tumors (p-value = 0.0097). There was no significant difference in tumor growth in the MDA-MB-231-LKB1+ASCs and MDA-MB-231-vector+ASCs groups. We compared differences in the number of lung and liver metastases in each group. We investigated stromal remodeling in tumors in each group. Our data show how the ECM alters cancer cell response, inducing LKB1 overexpressed cells to become more aggressive. The data presented here will help elucidate the cell-matrix interactions that are vital in migration and invasion in TNBC. Citation Format: Maryl Wright, Margarite Matossian, Madlin Alzoubi, Conner King, Steven Elliott, Melyssa Bratton, Elizabeth Martin, Bridgette Collins-Burow, Matthew Burow. Utilizing a decellularized PDX tumor matrix model to investigate LKB1 regulation of ECM remodeling in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 80.

Abstract C222: The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens.

Abstract Ewing sarcoma (EWS), the second most frequent bone cancer, is an aggressive primitive neuroectodermal tumor that occurs primarily in children, adolescents and young adults. A pathognomonic translocation involving the EWS gene on chromosome 22 and the ETS-type gene FLI1 on chromosome 11 occurs in about 85% of cases. The EWS/FLI1 fusion protein product of this translocation is a potent transcription factor that functions as an oncoprotein. The current study uses complementary approaches of high-throughput siRNA mediated RNAi and Investigational Agents screening to identify new therapeutic strategies for Ewing sarcoma. For RNAi screening the TC32 cell line, a well-characterized EWS cell line that expresses the EWS-FLI1 fusion transcript, was assayed for cell viability (CellTiterGlo, Promega) 72 hrs post siRNA transfection using a library of siRNAs corresponding to the genes encoding proteins with kinase function, three siRNA per gene (Ambion). Fifteen kinase genes were identified as inducing a significant decrease in the survival of TC32 cells when silenced by RNAi. These genes were chosen for validation in further EWS lines and two non-EWS cell lines. Using a cut off of a 40% or greater decrease in cell viability induced by two or more siRNAs per gene in at least at one EWS cell line eleven genes of interest were identified: PLK1, AURKB, AKT1, CDC2L1, CRKRS, ATP6VOB, CDC2L5, AURKA, CHEK1, NRBP2 and CDK4. The importance of several of these kinases in the survival of EWS cell lines was confirmed by an independent high-throughput screen of 63 human pediatric and adult sarcoma lines, including 19 EWS lines, with the Approved Oncology Drugs (AOD) library of compounds and an Investigational Agents library (350 agents) using inhibition of proliferation as an endpoint. Cells were exposed to compounds at varying concentrations (10μM to 1.5nM) for 96 h and the effect of compound on cell viability was monitored using Alamar Blue. The IC50 (50% growth inhibitory) concentrations of each agent on the cell lines were determined using curve fitting algorithms. Compared to the other sarcoma lines, the EWS lines were: more sensitive to eight Aurora kinase inhibitors (IC50 ∼0.05 µM) (IC50 ∼2.5 µM);more sensitive (IC50 ∼0.1 µM) to five checkpoint kinase (CHK) inhibitors (IC50 about 1.3 µM); and more sensitive to eight polo-like kinase (PLK1) inhibitors. These results demonstrate the power of integrating RNAi and compound inhibition studies to identify molecular targets for specific cancer types. We are currently further evaluating the inhibition of the mitosis-associated proteins identified by these complementary screening approaches as a strategy for the treatment EWS. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C222. Citation Format: Amy McCalla, Patrick Grohar, Scott Martin, Eugen Buehler, Suntae Kim, Natasha Caplen, Joel Morris, Mark Kunkel, David Evans, Anne Monks, Beverly Teicher, Lee Helman. The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C222.

Preparation of Recombinant Protein Spotted Arrays for Proteome‐Wide Identification of Kinase Targets

Protein microarrays allow unique approaches for interrogating global protein interaction networks. Protein arrays can be divided into two categories: antibody arrays and functional protein arrays. Antibody arrays consist of various antibodies and are appropriate for profiling protein abundance and modifications. Functional full-length protein arrays employ full-length proteins with various post-translational modifications. A key advantage of the latter is rapid parallel processing of large number of proteins for studying highly controlled biochemical activities, protein-protein interactions, protein-nucleic acid interactions, and protein-small molecule interactions. This unit presents a protocol for constructing functional yeast protein microarrays for global kinase substrate identification. This approach enables the rapid determination of protein interaction networks in yeast on a proteome-wide level. The same methodology can be readily applied to higher eukaryotic systems with careful consideration of overexpression strategy.