The Genetic Landscape Of FPD/AML Revealed CDC25C Mutation As a Driver That Promotes Malignant Transformation
Authors: Yoshimi Akihide , Toya Takashi , Nakagawa Masahiro , Kawazu Masahito , Nannya Yasuhito , Ichikawa Motoshi , Arai Shunya , Harada Hironori , Usuki Kensuke , Hayashi Yasuhide , Ito Etsuro , Kirito Keita , Nakajima Hideaki , Mano Hiroyuki , Kurokawa Mineo 
Published: October 2019

Abstract
Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. Although inherited RUNX1 mutation is the cause of thrombocytopenia, additional genetic events may be responsible for the tumor development as only 40 % of FPD/AML patients develop leukemia. Because of the rarity of this disorder, underlying mechanisms for malignant transformation in FPD/AML have not been elucidated. Thus we conducted a nationwide survey in Japan to collect samples and make a diagnosis of patients with familial thrombocytopenia or hematological malignancies. As a result, 56 pedigrees were extracted, and seven pedigrees with RUNX1 mutation were diagnosed as having FPD/AML, in which eight out of 14 patients had developed hematological malignancies. To systematically identify additional genetic alterations, we utilized whole exome sequencing in two patients with FPD/AML who had RUNX1_F303fsX566 mutation and developed MDS (Patient 1) or myelofibrosis (Patient 2) followed by AML. We identified 12 and 10 somatically acquired nonsynonymous mutations in these patients, respectively. Intriguingly, the two patients had common CDC25C mutation at codon 234 (D234G). Further genomic screening of other pedigrees revealed that four out of eight FPD/AML patients who developed hematological malignancy harbored somatic mutations in CDC25C (CDC25C_D234G in three patients and CDC25C_H437N in one patient). Recurrent CDC25C mutation in FPD/AML with subsequent hematological malignancy implies that it forms common genetic foundation of transformation in this disease. CDC25C is a phosphatase that prevents premature mitosis in response to DNA damage at the G2/M checkpoint, while it is constitutively phosphorylated at Ser216 throughout the interphase by c-TAK1. When phosphorylated at Ser216, CDC25C binds to 14-3-3 protein, which sequestrates CDC25C in the cytoplasm and inactivates it. In all of the mutated forms of CDC25C that we found in FPD/AML, their binding capacity with c-TAK1 and 14-3-3 protein was reduced, resulting in decreased phosphorylation status of CDC25C at Ser216. As a consequence, those CDC25C mutants led to enhanced mitosis entry, which was exaggerated by radiation-induced DNA damage. These results demonstrate that CDC25C mutation results in disruption of DNA checkpoint machinery. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and induces cell cycle arrest in hematopoietic cells, suggesting that the DNA checkpoint mechanism is activated in the presence of those types of RUNX1 mutation. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitosis 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. Interestingly, analysis of clonal evolution during leukemic transformation revealed that a clone defined by CDC25C mutation was dominant in the early phase of disease progression in both patients, which supports the idea that CDC25C mutation is associated with establishment of the founder clone during the leukemic progression of FPD/AML. In Patient 1, the founder clone with CDC25C mutation acquired FAM22G and COL9A1 (group 1) mutations, followed by occurrence of GATA2 and LPP (group 2) mutations to become a dominant clone in the AML phase, whereas another subclone of the founder defined by CHEK2 and DTX2 (group 3) mutations regressed. Similar hierarchical progression was observed in Patient 2. An additional single cell genomic sequencing of bone marrow cells from Patient 1 in the AML phase revealed that group 1/2 mutations and group 3 mutations were mutually exclusive, which supports our predicted model. Collectively, these results indicate that somatic mutation in CDC25C is a recurrent event in the early phase of leukemic progression of FPD/AML, which induces premature mitosis and genetic instability in hematopoietic cells with germline RUNX1 mutation.

Disclosures:
Usuki: Alexion Pharmaceuticals, Inc.: Speakers Bureau. Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.