Abstract 3439: Recurrent CDC25C mutations drive malignant transformation in FPD/AML
Authors: Yoshimi Akihide , Toya Takashi , Kawazu Masahito , Ueno Toshihide , Tsukamoto Ayato , Iizuka Hiromitsu , Nakagawa Masahiro , Nannya Yasuhito , Arai Shunya , Ichikawa Motoshi , Harada Hironori , Usuki Kensuke , Hayashi Yasuhide , Ito Etsuro , Kirito Keita , Nakajima Hideaki , Mano Hiroyuki , Kurokawa Mineo 
Published: May 2015

Abstract
Germline mutations in RUNX1 cause familial platelet disorder with predisposit ion to acute myelogenous leukemia (FPD/AML) which is characterized by platelet defects and hematological malignancies. The molecular pathogenesis is poorly understood because of the rarity of this disease, which has impeded the clinical improvement to refine cancer therapy and to identify patients at risk for the onset of malignancies in FPD/AML. Thus, we conducted a nationwide survey to identify patients with familial thrombocytopenia or hematological malignancies. After screening germline RUNX1 mutations in 73 index patients, 13 patients in 7 pedigrees were diagnosed as having FPD/AML, of which 7 had developed hematological malignancies. The mutational spectrum of FPD/AML was unveiled by whole-exome sequencing and targeted deep sequencing, and we identified CDC25C mutation as a recurrent target of FPD/AML, which was found in 7 of 13 (53 %) patients, including 4 FPD/AML patients who had not developed malignancies, although the variant allele frequencies were much lower than those found in patients with malignancies. Furthermore, GATA2 mutations were subclonal in 3 of 7 individuals with CDC25C abnormalities. Deep sequencing enabled an accurate estimation of allele frequencies for individual mutations and prediction of hierarchical architecture, which disclosed that CDC25C mutations form a founding pre-leukemic clone, followed by stepwise acquisition of subclonal mutations that contributed to leukemia progression. Single cell sequencing validated the estimation of the clonal evolution model. In all of the mutated forms of CDC25C, their binding capacity with c-TAK1 was reduced, resulting in decreased phosphorylation of CDC25C at Ser216. As a consequence, mutated CDC25C failed to bind 14-3-3 protein efficiently and stayed in the nucleus at G1/S phase. In accordance with these observations, CDC25C mutants enhanced mitosis entry, which was exaggerated by low dose radiation-induced DNA damage. These results suggest that CDC25C mutation results in disruption of the DNA checkpoint machinery, indicating that CDC25C is a novel target in human cancer. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and activates the DNA checkpoint mechanism. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitotic entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. In conclusion, malignant transformation of FPD/AML is formed by stepwise acquisition of mutations and clonal selection, which is initiated by a CDC25C mutation in the pre-leukemic phase and further driven by mutations in GATA2 or other genes. These findings will facilitate the diagnosis and monitoring of patients with FPD/AML who are at an increased risk of developing hematological malignancy.
Citation Format: Akihide Yoshimi, Takashi Toya, Masahito Kawazu, Toshihide Ueno, Ayato Tsukamoto, Hiromitsu Iizuka, Masahiro Nakagawa, Yasuhito Nannya, Shunya Arai, Motoshi Ichikawa, Hironori Harada, Kensuke Usuki, Yasuhide Hayashi, Etsuro Ito, Keita Kirito, Hideaki Nakajima, Hiroyuki Mano, Mineo Kurokawa. Recurrent CDC25C mutations drive malignant transformation in FPD/AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3439. doi:10.1158/1538-7445.AM2014-3439