Abstract 4964: Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia
Authors: Heikkinen Tuomas , Kämpjärvi Kati , Keskitalo Salla , Turunen Mikko , Kuusanmäki Heikki , Mäkinen Netta , Mustjoki Satu , Heckman Caroline , Varjosalo Markku , Vahteristo Pia 
Published: August 2015

Abstract
Mediator complex subunit 12 (MED12) is part of a Mediator, which is a large, highly conserved protein complex that participates in the regulation of general as well as gene-specific transcription. First somatic MED12 mutations were identified when exome sequencing revealed highly specific mutations in exon 2 in 70% of uterine leiomyomas studied. Additional mutations have subsequently been observed in exon 1, which further highlight the role of MED12 in these tumors. Functional analyses have indicated that exon 1 and exon 2 mutations lead to a diminished interaction between MED12 and other components of the Mediator kinase module, reduced Mediator-associated CDK kinase activity, and a unique global gene expression pattern. Recently, we observed similar mutations in chronic lymphocytic leukemia (CLL). Mutations were observed in 5% of the patients and the positive mutation status was associated with well-known poor prognosis markers in CLL. All the observed mutations both in uterine leiomyomas and CLL have been missense mutations or small insertions or deletions leading to an in-frame transcript.
Interestingly, the first MED12 mutation in a patient with T-cell acute lymphoblastic leukemia (T-ALL) was recently identified through exome sequencing. This is a nonsense mutation affecting the mutation hotspot observed in CLL. It is also the first nonsense mutation identified in the MED12 N-terminal mutation hotspot area in any tumor type. The patient has received bone marrow transplantation and is now in complete remission, but small amounts of cancer cells were collected before the treatment. RNA was extracted from the cells, and cDNA sequencing revealed that only the mutant allele is expressed. We have now generated a construct with the MED12 specific mutation and transfected it into HEK293 Flp-In cells. Wild-type MED12 is being used as a control. Western blotting with N- and C-terminal MED12 antibodies indicates that the mutant protein is being produced but that the N-terminal part is lacking.
We now aim to further characterize the function of this highly unusual mutation. Mutant and wild type protein will be produced for proteome-wide mass spectrometry analyses to detect possible effects on protein-protein interactions and also to gain more information on the translation start site. Also transcriptome-wide expression data will be produced to study the effect of the mutation on global gene expression profile. Overall, a mutation affecting the 5′ terminus of MED12 has now been identified in yet another cancer type, T-ALL. Our preliminary analyses indicate that the observed nonsense mutation escapes nonsense-mediated RNA decay and leads to a truncated protein product. Besides providing additional knowledge on the role of MED12 mutations in cancer, this exceptional mutation has potential to produce novel information on the normal MED12 function.
Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Salla Keskitalo, Mikko Turunen, Heikki Kuusanmäki, Netta Mäkinen, Satu Mustjoki, Caroline Heckman, Markku Varjosalo, Pia Vahteristo. Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia. [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 4964. doi:10.1158/1538-7445.AM2015-4964