In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 4687-4687
Abstract:
RAS mutations are found in around 30% of all human cancers, with KRAS being the most frequently activated RAS family of oncogenes. Although extensive efforts to develop attractive chemotherapeutic drugs targeting KRAS mutations with clinical benefit have been made, these experimental trials have often resulted in unsuccessful. Recently, we have successfully produced for the first time a novel alkylating agent (termed KR12) conjugated with the sequence-specific Pyrrole-Imidazole polyamide (PI polyamide), which was expected to have an ability to bind to base sequences of KRAS mutations at codon 12 (G12D and G12V). According to our results, KR12-treated colon cancer-derived LS180 cells carrying a KRAS G12D heterozygous mutation underwent remarkable G2/M cell cycle arrest, cellular senescence and subsequent p53-dependent apoptotic cell death in association with a massive down-regulation of mutant KRAS expression as examined by quantitative real-time RT-PCR and immunoblotting. In the present study, we have further assessed the sequence-specificity of KR12 in detail by using an in vitro gel mobility shift assay, binding affinity assays with surface plasmon resonance (Biacore system) and also determined IC50 of KR12 in a variety of colon cancer-derived cells with the distinct KRAS status. Gel mobility shift assay demonstrated that the mobility of the oligonucleotide containing KRAS mutation was significantly retarded in the presence of KR12 but not in the absence of KR12. In agreement, KR12 specifically bound to the oligonucleotide containing KRAS mutation with high affinity as examined by Biacore system in vitro. Notably, ligation-mediated PCR analysis (LM-PCR) revealed that KR12 indeed alkylate the adenine residue next to KRAS at codon G12D in LS180 cells. These observations imply that KR12 is capable to bind to and alkylate the expected KRAS sequence in vitro and in cells. Next, we have investigated the sensitivity to KR12 in a variety of colon cancer-derived cells. Based on our standard WST cell survival assay demonstrated that KRAS mutation-bearing SW480 (G12V), SW620 (G12V), and LS180 (G12D) cells exhibited a significantly higher sensitivity to KR12 as compared with HT-29 (WT), Caco-2 (WT), DLD-1 (G13D) and SW1463 (G12C) cells. Taken together, our present results strongly suggest that KR12 is a novel sequence-specific alkylating agent targeting KRAS G12D as well as G12V mutation, and thus might be a promising anti-cancer drug for the treatment of patients bearing malignant cancers with KRAS mutations. Citation Format: Kiriko Hiraoka, Takahiro Inoue, Hiroyuki Yoda, Atsushi Takatori, Takayoshi Watanabe, Nobuko Koshikawa, Toshinori Ozaki, Hiroki Nagase. A novel alkylating pyrrol-imidazole polyamide, KR12, specifically recognizes mutant KRAS genes and potently induces cell death. [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 4687. doi:10.1158/1538-7445.AM2015-4687
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2015-4687
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2015
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
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