Abstract 2341: Sleeping Beauty transposon mutagenesis screen identifies cancer genes of uterine leiomyosarcoma driving sarcomagenesis and lung metastasis
Authors: Michiko Kodama, Takahiro Kodama, Justin Y. Newberg, Jean C. Tien, Roberto Rangel, Aya Nakae, Kae Hashimoto, Seiji Mabuchi, Kenjiro Sawada, Tadashi Kimura, Nancy A. Jenkins, Neal G. Copeland
Journal: Cancer Research (2018)

Abstract Introduction Uterine leiomyosarcoma (ut-LMS) is one of the most aggressive malignancies with extremely poor prognosis. The 5-year survival rate is 0-20% for patients with tumors beyond uterus and 50% for even early stage patients due to high hematological metastatic potential. Because of its rarity, large-scale genetic profiling could not be done and thus its molecular mechanisms remain largely unknown. To solve this problem, we conducted in vivo sleeping beauty (SB) transposon mutagenesis screen and identified the key genetic drivers of ut-LMS. Methods We crossed Amhr2-Cre knock-in mice, transposon transgenic mice (T2Onc2), SB transposase knock-in mice (Rosa-LSL-SBase), Pten floxed mice, and Kras G12D knock-in mice, generating experimental mice (Amhr2-Cre Tg/+;T2Onc2 Tg/+;SBase KI/+;Pten fl/fl;Kras LSL-G12D/+) and control mice (Amhr2-Cre Tg/+; Pten fl/fl;Kras LSL-G12D/+). The experimental mice have homozygous deletion of Pten, constitutive activation of Kras and SB transposon mobilization in uterine smooth muscle cells. Fifty-nine experimental mice and 14 control mice were aged until moribund. Results All of the experimental mice died of multiple uterine tumors by 2 months of age, while none of the control mice developed uterine tumors. These uterine tumors were histologically diagnosed as ut-LMS by their morphology and immunohistochemical positivity of desmin and alpha-SMA. These results clearly indicated that transposon mutagenesis was required for sarcomagenesis of ut-LMS. To identify genes mutagenized by SB transposon in these tumors, we performed splink HiSeq sequence of gDNA from 80 primary uterine tumors and 24 normal uterus of mice with uterine tumors. Gene-centric common insertion site analysis (gCIS) identified 19 CIS genes in primary uterine tumors but no CIS genes in normal uterus, indicating the selective enrichment of transposon insertions in cancer genes in tumors. By further focusing on the transposon insertions with high sequence read counts, we identified trunk mutations driving sarcomagenesis that include inactivating mutations of NF1 and activating mutations of one of zinc fingers gene family. Its inhibition via siRNA in human ut-LMS cell lines, SK-LMS1, SK-UT1, and SKN, impaired cell proliferation and sphere formation, suggesting the importance of this gene for the survival of ut-LMS. Next, to identify the metastatic drivers of ut-LMS, we induced the lung metastasis by tail vein injection of 9 cell lines established from SB uterine tumors. After the sequential in vivo passage of metastatic SB tumors, we have obtained 50 lung metastatic tumors in immunocompetent mice. Sequencing and gCIS analysis of 50 lung metastases identified 3 potential driver genes of lung metastases. Conclusion SB mutagenesis screen discovers multiple cancer genes of ut-LMS driving sarcomagenesis and lung metastasis. Citation Format: Michiko Kodama, Takahiro Kodama, Justin Y. Newberg, Jean C. Tien, Roberto Rangel, Aya Nakae, Kae Hashimoto, Seiji Mabuchi, Kenjiro Sawada, Tadashi Kimura, Nancy A. Jenkins, Neal G. Copeland. Sleeping Beauty transposon mutagenesis screen identifies cancer genes of uterine leiomyosarcoma driving sarcomagenesis and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2341.