Abstract 3176: Recurrent SETBP1 mutations in atypical chronic myeloid leukemia abrogate an ubiquitination site and dysregulate SETBP1 protein levels.
Authors: Piazza Rocco , Valletta Simona , Winkelmann Nils , Redaelli Sara , Spinelli Roberta , Pirola Alessandra , Antolini Laura , Mologni Luca , Donadoni Carla , Papaemmanuil Elli , Schnittger Susanne , Dong-Wook Kim , Boultwood Jacqueline , Rossi Fabio , Gaipa Giuseppe , De Martini Greta , di Celle Paola Francia , Jang Hyun Gyung , Fantin Valeria , Bignell Graham R. , Magistroni Vera , Haferlach Torsten , Pogliani Enrico Maria , Campbell Peter , Chase Andrew J. , Tapper William J. , Cross Nick C.P. , Gambacorti Passerini Carlo 
Published: August 2014

Abstract
Atypical Chronic Myeloid Leukemia (aCML) shares clinical and laboratory features with Chronic Myeloid Leukemia (CML), but it lacks the pathognomonic BCR-ABL1 fusion. The molecular pathogenesis of this disease has remained elusive and the outcome dismal. To investigate the molecular pathogenesis of aCML, we applied exome sequencing and RNA-SEQ to aCML, with the aim of identifying novel recurrent driver mutations. Whole-exome sequencing of 9 aCML patients(pts) revealed the presence of 70 unique mutations, including recurrent alterations of SETBP1 in 3 cases: 2 cases with a G870S and a case with a D868N alteration were found. Targeted resequencing in 70 aCMLs, 574 pts with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17/70 aCML (24.3%; 95% CI: 16-35%), 4/30 (13%) MDS/MPN-u, 3/82 (3.6%) CMML and 0/100 MDS. aCML pts with SETBP1 mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. The SETBP1 gene encodes for a predominantly nuclear protein with a predicted MW of 170kDa. Germline mutations of SETBP1 were previously described in pts affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities and neuroepithelial neoplasms. The vast majority of aCML SETBP1 mutations (85%) was located between residues 858 and 871 and were identical to the germline changes seen in pts with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif identified a putative functional site (aa. 868-873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. The prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859-879): while the wild type (wt) peptide could efficiently bind beta-TrCP, a peptide presenting G870S was incapable of binding this subunit, indicating a possible alteration in SETBP1 protein stability caused by the mutation. In line with the known binding model of beta-TrCP, dephosphorylated peptides failed to bind beta-TrCP, therefore confirming the specificity of the interaction. In agreement with these findings, TF1 cells transduced with SETBP1 G870S showed increased SETBP1 and SET protein levels, decreased PP2A activity and increased proliferation rates when compared to cells expressing the wt SETBP1 gene. Mutated SETBP1 represents a novel oncogene specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1.
Citation Format: Rocco Piazza, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Kim Dong-Wook, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R. Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter Campbell, Andrew J. Chase, William J. Tapper, Nick C.P. Cross, Carlo Gambacorti Passerini. Recurrent SETBP1 mutations in atypical chronic myeloid leukemia abrogate an ubiquitination site and dysregulate SETBP1 protein levels. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2013-3176