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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. © 2015 by The American Society of Hematology.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) is a subset of Non-Hodgkin Lymphoma that positively took advantage of crizotinib development, a selective ALK inhibitor. Crizotinib is able to block ALK kinase activity which is fundamental for cancer cell survival. Constitutively active ALK kinase triggers the activation of several pro-proliferative and pro-survival downstream pathways, such as PI3K/AKT/mTOR. Despite the positive impact of targeted therapy on ALCL treatment, resistant clones tend to be selected during therapy. Strategies to overcome resistance include the design of more potent and selective drugs and the use of combined therapy approaches that allow for the simultaneous targeting of more than one node essential for cancer cells survival. Following this strategy, we decided to investigate the effects of combined ALK/mTOR inhibition. We first observed a synergistic effect of the combination of ALK inhibitors (crizotinib, alectinib or PF-3922) with an mTOR inhibitor (temsirolimus) in proliferation assays. Interestingly, the synergistic effect was observed only in ALK+ cell lines (Karpas 299, SUDH-L1 and SUPM2), while no synergism was observed in ALK- cell line (U937) as well as in normal lymphocytes. The Combination Index (CI) ranged from 0.16 to 0.45 indicating a synergistic/strong synergistic effect, accordingly to Chou classification. (Chou, 2006) (Table 1). The positive cooperation resulted in an increased inhibition of mTOR effectors p70S6K, 4EBP1 and eIF4B compared to single agent treatment as observed in immunoblot analysis performed on Karpas 299 treated for 4 hours. At the cell cycle level, the use of the drugs in combination induced a sharp block in G0/G1 phase, as observed by propidium iodide analysis performed on Karpas 299 at 48-72 and 96 hours. Long term exposure of ALK+ cells to either alectinib or temsirolimus, led to the establishment of resistant cell lines, while the exposure to the combination prevented the selection of resistant clones. Interestingly, the cell line resistant to alectinib showed a marked increase of ALK both at mRNA and protein level. In vivo, nude mice were injected with Karpas 299 cells. As the tumor masses reached 150mm3, mice were randomized and treated for 12 days with the combination of PF-3922 (0.5 mg/kg, administered per os twice a day) and temsirolimus (i.p., 1 mg/kg every other day) or with the single agents. The combined treatment induced a faster regression of the tumor masses compared to the single agents-treated mice. Moreover, responses were sustained for a longer period of time, upon treatment stop. (Figure 1) In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for patients affected by ALK+ ALCL. Table 1. combination indexes from proliferation experiments. Synergism levels calculated accordingly to Chou, Pharmacological reviews, 2006 Crizotinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:1 0.42 0.35 0.33 0.37 Synergism SUDHL1 3:1 0.49 0.45 0.42 0.45 Synergism SUPM2 1:1 0.59 0.33 0.29 0.40 Synergism NPM-ALK- U937 1:1 >10 >10 >10 >10 Antagonism HD Lymphocytes 1:1 >10 >10 >10 >10 Antagonism Alectinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:3 0.56 0.29 0.15 0.33 Synergism SUDHL1 1:3 0.53 0.45 0.38 0.45 Synergism SUPM2 1:10 0.65 0.18 0.06 0.3 Strong synergism NPM-ALK- U937 1:3 1.5 >10 >10 >10 Antagonism HD Lymphocytes 1:3 1.2 2.7 7.2 3.7 Antagonism PF06463922 - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:10 0.30 0.15 0.15 0.20 Strong synergism SUDHL1 1:30 0.57 0.41 0.29 0.42 Synergism SUPM2 1:3 0.30 0.12 0.06 0.16 Strong synergism NPM-ALK- U937 1:10 3.2 5.39 >10 >10 Antagonism HD Lymphocytes 1:3 1.4 8.8 >10 >10 Antagonism Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Disclosures No relevant conflicts of interest to declare.

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel–Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel–Giedion syndrome caused by SETBP1 mutations., SETBP1 variants occur as somatic mutations in several malignancies and as de novo germline mutations in developmental disorders. Here the authors provide evidence that SETBP1 binds to gDNA in AT-rich promoter regions to promote target gene upregulation, indicating SETBP1 functions directly to regulate transcription.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/ mTOR, indispensable for survival of ALK-driven tumors. Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while longterm exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

In the past years, the improvements in sequencing technology led to the development of “Next Generation Sequencing” (NGS) technologies. Several NGS approaches exist. Whole genome sequencing (WGS) and whole exome sequencing (WES) allow the identification of genomic alterations such as small insertions/deletions, point mutations and structural variants. Whole transcriptome sequencing (RNA-Seq) permits to quantify gene expression profiles and to detect alternative splicing and fusion transcripts. Recently, by using WES on atypical chronic myeloid leukemia (aCML) samples, our group identified recurrent mutations in SETBP1 gene; also, by using RNA-Seq on acute myeloid leukemia (AML), we identified a new fusion gene: ETS2-ERG. In aCML, SETBP1 mutations disrupt a degron binding site, leading to a decreased protein degradation. This leads to an increased amount of SETBP1 protein interacting with its natural ligand SET, which in turn acts inhibiting the protein phosphatase 2A (PP2A) oncosuppressor. Interestingly, the SETBP1 mutational cluster affected in aCML is highly conserved and the same mutations were also observed in the Schinzel-Giedion syndrome (SGS). However, the inhibition of the PP2A by SET, the only known interactor of SETBP1, does not explain the phenotype of SGS. To further characterize the role of SETBP1 protein, 293 Flp-In isogenic cellular models expressing the empty vector or the wild type (WT) or mutated (G870S) form of SETBP1 were established. In these models SETBP1 was fused with a V5 tag. Chromatin Immunoprecipitation sequencing experiments (Chip-Seq) performed against V5 confirmed the binding of SETBP1 to DNA, both for the WT and G870S forms. In addition, RNA-Seq experiments were performed. The comparison between Chip-Seq and RNA-Seq data has allowed us to identify 130 genes presenting both the binding of SETBP1 to their promoter region and transcriptional upregulation. Together these data suggest a role for SETBP1 as a transcriptional activator. Co-immunoprecipitation (Co-IP) experiments in transiently transfected HEK293T cells coupled with mass spectrometry (MS) analysis were performed to identify potential interactors of SETBP1. MS analysis led to the identification of the host cell factor 1 (HCF1), a component of the SET1/KMT2A COMPASS-like complex. Independent validation by western blot and fluorescence resonance energy transfer (FRET) confirmed the direct binding of HCF1 to SETBP1. Further independent experiments confirmed the Co-Ip of SET1/KMT2A and PHF8 with SETBP1. SET1/KMT2A is a core component of COMPASS-like complex and possesses H3K4 methyltransferase activity, whereas PHF8 possesses H4K20 demethylase activity. Both marks are associated with actively transcribed genes. Taken together, we have shown that SETBP1 protein is able to act as a transcriptional activator recruiting the HCF1/KMT2A/PHF8 complex. In a previous study, comparing cytogenetic analysis and RNA-Seq to detect chromosomal abnormalities on AML patient samples, a new fusion between the ETS2 and ERG genes was reported. The patient carrying this fusion was affected by acute promyelocytic leukemia (APL) and did not respond to therapy with retinoic acid. The role of the ETS2-ERG fusion is not known. To gain insight about the functional role of ETS2-ERG fusion in APL two cellular models were established. HL-60 and NB-4 cells were stable transfected with retroviral empty vector or with a vector carrying the fusion gene. This vector also carries the GFP as a positive selection marker. HL-60 cells carrying the ETS2-ERG fusion treated with retinoic acid showed a decrease in the expression at membrane level of the differentiation marker CD11b. This suggests that the ETS2-ERG fusion is able to impair the differentiation of APL cells upon retinoic acid treatment. Further experiments are ongoing to confirm the data in the NB4 cellular model.

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.

Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the Myeloproliferative/Myelodysplastic (MPN/MDS) group. The molecular lesions responsible for the onset of aCML remained unknown until 2013 when recurrent somatic mutations of SETBP1 were identified. However, the frequency of SETBP1 mutations in aCML does not exceed 25-30%, which suggests that other lesions may play a role in the remaining cases. To gain further insight into the somatic variants responsible for the onset of aCML, we generated whole-exome and transcriptome sequencing data on 15 matched case/control aCML samples. A total of 151 non-synonymous and 42 synonymous single-nucleotide somatic variants were identified. Of these, 140 were transitions and 53 transversions. Of the non-synonymous mutations, 141 were missense and 10 nonsense mutations. In 2/15 (13.3%) samples we identified the presence of missense, single-nucleotide somatic variants occurring in the ETNK1 gene affecting two contiguous residues: H243Y and N244S. Sanger sequencing confirmed the presence and the somatic nature of the variants. Targeted resequencing of 383 clonal hematological disorders showed evidence of mutated ETNK1 in 7/70 aCML (10.0%, 95% C.I. 4.6-19.5%) and in 2/77 chronic myelomonocytic leukemia samples (CMML; 2.6%, 95% C.I. 0.2-9.5%) %), while no ETNK1 mutations were identified in the remaining hematological disorders. All the variants were heterozygous and clustered in the same, highly conserved region within the kinase domain (1/9 H243Y and 8/9 N244S). Somatic, heterozygous ETNK1 variants have been also recently reported in 10% of Systemic Mastocytosis (SM) cases and in 22% of SM with associated hypereosinophilia (Lasho T et al., Abstract 4062, EHA2014); strikingly, there is a large overlap between the variants that we identified in aCML and CMML and those described for SM (3 N244S and 2 G245A), which suggests that the common hotspot region may play a critical and yet unknown functional role. The hitherto described data suggest that ETNK1 variants are restricted to a limited subset of hematological disorders. This is further supported by the lack of somatic ETNK1 mutations in 60 paired whole-genome and over 600 exomes, comprising 276 paired tumor/germline primary samples and 344 cancer cell lines (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). In 2/6 ETNK1 mutated aCML cases (33%, 95% C.I. 9%-70%), we detected the presence of a coexisting somatic SETBP1 variant. The fraction of SETBP1 mutations identified in this group is perfectly in line with the overall frequency of SETBP1 mutations in aCML, suggesting that mutations occurring in ETNK1 and SETBP1 are not mutually exclusive. To discriminate if ETNK1 and SETBP1 mutations occur in different or in the same clone, we performed colony assay experiments, revealing the coexistence of the two somatic mutations within the same clone. Liquid Chromatography – Mass Spectrometry experiments revealed that in ETNK1 mutated cells the intracellular levels of phosphoethanolamine are over 5-fold lower than in the wild-type counterpart (p < 0.05), suggesting that ETNK1 mutations may impair the physiological catalytic activity of the kinase. Taken globally these data identify ETNK1 somatic mutations as a new oncogenic lesion in aCML and CMML, two overlapping MDS/MPN neoplasms. They also show that ETNK1 variants apparently cause a loss-of-function effect, leading to a decrease in the intracellular levels of phosphoethanolamine. Disclosures Campbell: 14M Genomics Limited: Consultancy, Equity Ownership.