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3701 PTK7 Is Involved in the Chemotherapy Resistance of T-Cell Acute Lymphoblastic Leukemia through Interaction with FADD-Death Domain

Molecular Pharmacology, Drug Resistance – Lymphoid and Other Diseases
Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology, Drug Resistance – Lymphoid and Other Diseases: Poster III
Monday, December 7, 2015, 6:00 PM-8:00 PM
Hall A, Level 2 (Orange County Convention Center)

Hideki Goto, MD, PhD1,2*, Rachel Wilkie Grantham, PhD1*, Motoaki Mizuuchi3*, Tetsuji Yamaguchi3*, Takanori Teshima, MD, PhD2, John C. Reed, MD, PhD1 and Shu-ichi Matsuzawa, PhD1*

1Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA
2Department of Hematology, Hokkaido University Graduate School of Medicine, Sapporo, Japan
3JSR micro Inc., La Jolla, CA

Apoptosis-based therapies have been used as a powerful weapon against hematologic malignancies (Reed JC et al, Blood 2005 15; 408-418). One strategy for attacking cancer is to overcome its resistance to apoptosis via specific targeting molecules. In this study, we are focusing on protein tyrosine kinase 7 (PTK7) as a new target for cancer therapy. PTK7, also known as CCK4, is a member of transmembrane tyrosine kinase family proteins that has been implicated in cell migration, polarity and apoptosis. PTK7 is overexpressed in acute T-cell lymphoblastic leukemia cells, and a recent report indicated that PTK7 expression was associated with poor prognosis. However, the mechanism by which PTK7 elicits its anti-apoptotic effects and directly promotes tumor proliferation has not yet been clarified. In addition, no data has been reported regarding the relationship between doxorubicin resistance and PTK7 expression. In this study, we investigated the role of PTK7 in chemotherapy resistance. Two different shRNA lentiviruses targeting PTK7 were stably infected in Jurkat cells (Jurkat tet-shPTK7#1 and #2), and gene knockdown assays against PTK7 were performed. In the presence of doxycycline, we observed a decrease in the viability of PTK7 knockdown cells following stimulation with αFAS-Ab (Jurkat tet-shPTK7#1: 28.6% ± 5.2%, #2: 8.7% ± 0.3%) compared to control cells (considered to be 100%). Similar results of decreased cell viability were also obtained with TRAIL stimulation (Jurkat tet-shPTK7#1: 22.5% ± 5.9%, #2: 14.7% ± 9.8%) compared to control cells (considered to be 100%). Furthermore, we observed a decrease in the viability of PTK7 knockdown cells following treatment with Doxorubicin compared to control cells treated with scramble siRNA. The reduction of cell viability in PTK7 knockdown cells could not be attributed to PTK7 knockdown itself since annexin-V and PI staining showed limited apoptotic changes in Jurkat tet-shPTK7 cells. These changes were also not the result of Fas receptor (CD95) and Trail receptor (DR4, DR5) expression up-regulation as flow cytometric analysis revealed no differences in the expression rates of Fas receptor and Trail receptor between scramble control cells and PTK7 knockdown cells. Very interestingly, PTK7 knockdown had no effect on viability when non-cancer cell lines were stimulated with Fas, TRAIL and Doxorubicin, suggesting that the synergistic effect of PTK7 knockdown and TNF family cytokine treatment is cancer specific. Next, to identify interaction partners of the PTK7 cytoplasmic domain (PTK7-cyto), we performed a yeast two-hybrid assay, and showed that FADD-DD was the only molecule found to bind to PTK7-cyto in both LEU2 reporter and LacZreporter assays. This protein interaction was further verified by co-immunoprecipitation assays. Finally, to demonstrate the activation of Caspase-8 and PARP, which is induced by TNF-family death receptors, immunoblotting was performed. The shRNA-mediated silencing of PTK7 in Jurkat cells indeed increased Fas- and TRAIL-induced processing of caspase-8 and PARP compared to control RNA. These data suggest that PTK7 appears to be important for a proximal step in Fas and TRAIL signaling, which is consistent with the selective sensitization of tumor cells to the extrinsic apoptosis pathway. Together, our findings reveal an unexpected role for PTK7 in terms of death receptor-mediated apoptosis, suggesting that PTK7 could provide a new target for cancer therapy.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH