Recurrent SETBP1 Mutations in Atypical Chronic Myeloid Leukemia Abrogate an Ubiquitination Site and Dysregulate SETBP1 Protein Levels

Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 170 kD. Germline mutations of SETBP1 were described in patients affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities, and presenting neuroepithelial neoplasms. In an effort to investigate the molecular pathogenesis of myeloid malignancies we applied a HTS strategy, including both exome sequencing and RNA-SEQ, to atypical Chronic Myeloid Leukemia (aCML), as defined by WHO criteria, with the aim of identifying novel recurrent driver mutations. aCML shares clinical and laboratory features with CML, but it lacks the pathognomonic BCR-ABL1fusion. Since no specific recurrent genomic or karyotypic abnormalities have been identified in aCML, the molecular pathogenesis of this disease has remained elusive and the outcome dismal (median survival 37 months) with no improvement over the last 20 years. This sharply contrasts with the outcome for CML, for which the prognosis was dramatically improved by the development of imatinib as a specific inhibitor of the BCR/ABL protein. Whole-exome sequencing of 9 aCML patients revealed the presence of 62 unique mutations (range 5–14 per patient), including a recurrent alteration of SETBP1 (G870S and D868N) in three cases. Targeted resequencing performed in 70 aCMLs, 574 patients with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17 of 70 aCML patients (24.3%; 95% CI: 16–35%), 4 of 30 (13%) MDS/MPN-u and 3 of 82 (3.6%) CMML patients. Patients with mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. TF1 cells transfected with SETBP1G870S showed increased SET levels, decreased PP2A activity and increased proliferation rates. The vast majority of mutations (85%) was located between residues 858 and 871, in the SKI homologous region of SETBP1, and were identical to germline changes seen in patients with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif (ELM) identified with high probability score a putative functional site (aa. 868–873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. This prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859–879): while the wild type peptide could efficiently bind beta-TrCP as predicted, a peptide presenting the G870S mutation was incapable of binding this E3 ligase subunit, indicating a possible alteration in SETBP1 protein stability caused by this mutation. In agreement with these findings, cells transfected with SETBP1G870Sshowed increased levels of SETBP1 protein when compared to cells with similar expression levels of the wild type gene. Finally, RNA-SEQ yielded gene expression profiles with overrepresentation of genes under the control of Transforming Growth Factor Beta 1 (TGFβ1) among genes differentially expressed between SETBP1-mutated and unmutated aCML patients. Mutated SETBP1 represents a novel type of oncogene which is 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. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.