Carla Donadoni

Recurrent SETBP1 Mutations in Atypical Chronic Myeloid Leukemia

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16–35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.

Synthesis and biological evaluation of benzo(4,5)imidazo(1,2-c) pyrimidine and benzo(4,5)imidazo(1,2-a)pyrazine derivatives as anaplastic lymphoma kinase inhibitors

Chromosomal translocations involving anaplastic lymphoma kinase (ALK) are the driving mutations for a range of cancers and ALK is thus considered an attractive therapeutic target. We synthesized a series of functionalized benzo[4,5]imidazo[1,2-c]pyrimidines and benzo[4,5]imidazo[1,2-a]pyrazines by an aza-Graebe-Ullman reaction, followed by palladium-catalyzed cross-coupling reactions. A sequential regioselective cross-coupling route is reported for the synthesis of unsymmetrically disubstituted benzo[4,5]imidazo[1,2-a]pyrazines. The inhibition of ALK was evaluated and compound 19 in particular showed good activity against both the wild type and crizotinib-resistant L1196M mutant in vitro and in ALK-transfected BaF3 cells.

Abstract 2993: Patterns of recurrent mutations in SETBP1 mutated and wild-type atypical Chronic Myeloid Leukemia patients.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Atypical Chronic Myeloid Leukemia (aCML) is a heterogeneous disorder belonging to the group of myelodysplastic/myeloproliferative (MDS/MPN) syndromes. The molecular pathogenesis of this disease is still unclear and the outcome is poor with no improvement over the last 20 years. We applied whole exome sequencing approach in 9 aCML patient samples in order to identify possible recurrent alterations. The analysis revealed the presence of unique mutations in 70 genes with 3 cases of SETBP1 alterations. Some of the genes identified as mutated in the initial set of 9 patients (IDH2, MTA2, EPHB3, ETNK1, GATA2, IRAK4) and having a score higher than 1 in the oncogenic GeneRanker database were resequenced in a cohort of 40 aCML patients (15 with and 25 without SETBP1 mutations). With the exception of IDH2, no other gene was found mutated in any case apart from the index patient. Evaluation on a larger cohort of 70 aCML samples revealed recurrent SETBP1 mutations in 24.3% of cases (see designated abstract). To test the relationship between SETBP1 variants and mutations in oncogenes known to be involved in myeloid malignancies, mutations in ASXL1, CBL, CEBPA, DNMT3A, EED, EZH2, FLT3, IDH1/2, JAK2, JARID2, NPM1, N/KRAS, RBBP4, RUNX1, SF3B1, SUZ12, TET2 and WT1 were evaluated in a population of 61 aCML patients (14 with and 47 without SETBP1 mutations) by Sanger sequencing. Overall we identified 60 mutations in 14 genes: 28 were missense point mutations, 13 nonsense point mutations, 15 missense ins/del and 4 ins/del leading to a premature stop codon. No mutations were found in IDH1, RBBP4, NPM1, JAK2, FLT3, DNMT3A. Mutations in ASXL1 were present in 14 patients and appeared more frequent in patients with mutated SETBP1 (36% vs 19%) while the 15 TET2 mutations were more prevalent in patients with SETBP1 WT than in mutated samples(28% vs. 14%). Further associations will be presented at the meeting, although further analysis on larger cohorts of patients will be necessary to determine the significance of this differences. Additional data on epigenetic signature of aCML will clarify the role of epigenetic dysregulation in aCML and related diseases. Citation Format: Sara Redaelli, Simona Valletta, Rocco Piazza, Nils Winkelmann, 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. Patterns of recurrent mutations in SETBP1 mutated and wild-type atypical Chronic Myeloid Leukemia patients. [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 2993. doi:10.1158/1538-7445.AM2013-2993

Abstract 3176: Recurrent SETBP1 mutations in atypical chronic myeloid leukemia abrogate an ubiquitination site and dysregulate SETBP1 protein levels.

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