PO-030 Functional analysis of mastl mutations in cancer

Introduction Mastl, also known as Greatwall, is a protein kinase essential for proper chromosome condensation and progression through mitosis and meiosis. It belongs to the AGC kinase family and, particularly, presents a non-conserved insertion of 550 aa at the corresponding T-activation loop site in the C-lobe (usually 20–30 aa). This non-conserved middle region (NCMR) is not considered to have an essential role for Mastl activity but its function in unknown. Mastl is involved in the inhibition of protein phosphatase 2A (PP2A)-B55 complexes to maintain the mitotic state. By using a conditional knockout in mouse generated in our lab, it was shown that mammalian Mastl is essential for mouse embryonic development and cell cycle progression. Mastl was initially found in humans as a gene mutated in thrombocytopenia and preliminary data suggests its overexpression in tumours. However, very little is known about this protein in human disease. Material and methods Genomic data from repositories of cancer somatic mutations include MASTL NCMR indel mutations, leading to the generation of a truncated protein. Exome sequencing studies in Mastl in gastric cancers show that mutant tumours present microsatellite instability (MSI). We have studied the prevalence of these mutations by sequencing MASTL in a subset of samples from colon and stomach patients. To evaluate the therapeutic relevance of this kinase, functional assays have been performed, as complementation studies and kinase assays. To mimic the cancer mutations we have generated a new mouse model using CRISPR/Cas9 technology. We are currently performing several assays such as focus assays, scratch assays, soft agar and colony formation on cells derived from this model. In addition, we are using a chemical-induced colorectal carcinoma model to study the role of these mutant kinases in cancer. Results and discussions A heterozygous exonic indel mutation has been found in an MSI +CRC from the 21 patient samples sequenced. Functional assays with the mutant enzyme resulted in a partial rescue in DNA segregation observed in Mastl-null cells. Mastl mutant forms resulted in embryonic lethality in homozygosis. Therefore, our carcinogenesis models are performed in heterozygous mice, thus mimicking cancer mutations. Conclusion Mastl indel mutations in the NCMR region lead to the expression of truncated shorter forms. These Mastl mutated forms are not able to fully accomplish the role of Mastl in mitosis. Mutant heterozygous mice, mimicking MASTL cancer mutations, are viable and fertile.