Abstract 489: Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo
Authors: Ouchi Kazutaka , Kuwahara Yasumichi , Iehara Tomoko , Konishi Eiichi , Hosoi Hajime 
Published: August 2015

Background: We previously showed that INI1/SNF5 reexpression increased transcriptional activity at the NOXA locus in malignant rhabdoid tumor (MRT) cell lines resulting in NOXA protein production. In other cancer cells, the binding between NOXA and MCL-1 induces apoptosis after exposure to chemotherapeutic agents. Therefore, we hypothesized that loss of NOXA alters the apoptotic pathway in MRTs and contributes to their resistance to chemotherapeutic agents.

Methods: The TTC549 and KP-MRT-RY MRT cell lines were transfected with the pcDNA 3.1(+)-wild type hNOXA plasmid, a kind gift from Dr. Kelekar, University of Minnesota, using Lipofectamine reagent and then selected with G418. To generate MCL-1 knockdown MRT cell lines, we transfected a small interfering (si) RNA targeting MCL-1 using Lipofectamine reagent. Cells were plated in growth medium for 24 hrs and then treated with serial dilutions of doxorubicin (DOX) and/or TW-37. To generate mice xenografts for both TTC549 MCL-1 stable knockdown (KD) and control cell lines, we established two independently derived MCL-1 stable knockdown TTC549 cell lines using lentiviral vectors encoding a shRNA targeting MCL-1 and a negative control cell line (pLKO.1). We injected either 5 × 106 TTC549 MCL-1 KD or control cell lines s.c. into athymic mice. One week after injection, mice were treated once daily with DOX (15mg/kg) or vehicle alone for five days.

Results: In both NOXA expressing and MCL-1 KD MRT cells, we observed a dramatic reduction of the IC50 values of DOX treatment. Moreover, apoptosis was also observed at 48 hrs after DOX treatment at concentrations less than the IC50 values for control cells. Immunoprecipitation of MCL-1 showed that expressed NOXA associated with MCL-1 in MRT cells. Furthermore, we observed that mean tumor volume in the DOX-treated MCL-1 KD group was significantly smaller than that in the vehicle-treated MCL-1 knockdown group. Next, we assessed the effects of TW-37, an inhibitor that binds to the Bcl-2 homology domain 3 (BH3) of proteins including MCL-1, in combination with DOX. Using the combination index, we found that DOX and TW-37 displayed modest synergy in both MRT cell lines (CI&lt;1) in vitro and in vivo.

Discussion: Ectopic expression of NOXA or knockdown of MCL-1 increased DOX sensitivity in MRT cell lines. These results indicate that loss of INI1/SNF5-mediated regulation of NOXA expression impairs an apoptotic pathway leading to repression of MCL-1 followed by the resistance to chemotherapeutic agents. We also found that TW-37, a BH3 only protein mimetic, has a synergistic effect with DOX on MRT cell lines in vitro and in vivo, providing a potential foundation for molecular targeting therapy for MRT.

Citation Format: Kazutaka Ouchi, Yasumichi Kuwahara, Tomoko Iehara, Eiichi Konishi, Hajime Hosoi. Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2015-489