Academic literature on the topic 'ETNK1'

Abstract 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.

ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p&lt;0.0001). Since ROS are responsible for DNA oxidative damage, we firstly generated ChIP-Seq data for ETNK1 mutated cells using an antibody raised against the oxoguanine (oxoG) and we compared oxoG signal against the wild-type cell line, to assess whether ETNK1 mutations could cause accumulation of DNA lesions. This analysis revealed a significant increase in oxoG in mutated cells, compared to WT (p=0.018). Then, we investigated if these lesions were driving the onset of a mutator phenotype by applying the 6-thioguanine (6-TG) resistance assays to our cell models, showing that in the mutated cells there was a 5.4 fold increase in colony number compared to the WT line (p&lt;0.0001). Moreover, we investigated if the ROS-mediated genotoxic insult operating in ETNK1-mutated lines could be also associated with an increase in DNA double-strand breaks. Comparison of ETNK1-N244S and ETNK1-WT lines revealed a sharp increase in the number of γH2AX foci (2.52 fold increase; p=0.0002) in the former. At this point, we hypothesized that the decreased P-Et concentration in ETNK1-mutated cells could be responsible for the increased mitochondrial activity. ETNK1-N244S cells treated with P-Et showed a complete restoration of the normal mitochondrial membrane potential and generation of ROS. Moreover, the mutator phenotype was reverted by P-Et treatment, supporting the hypothesis of a direct involvement of P-Et in the induction of DNA damage. To dissect the mechanism by which P-Et intracellular levels were able to control mitochondria activity, we isolated the mitochondrial oxidative phosphorylation complexes I to IV and we measured the activity of each complex in absence/presence of increasing P-Et concentrations. This approach revealed a profound, dose-dependent decrease in redox activity for mitochondrial complex II (P-Et 10μM: 1.80 fold decrease; p=0.0012; P-Et 20μM: 7.40 fold decrease; p&lt;0.0001; P-Et 50μM: 28.85 fold decrease; p&lt;0.0001) and virtually no effect for the other three complexes, indicating that P-Et controls mitochondria potential through direct inhibition of complex II. To gain insight into the specific mechanism by which P-Et could repress complex II, we analyzed its activity in competition assays in presence of both P-Et and increasing concentration of succinate, the endogenous substrate of succinate dehydrogenase (SDH), showing that succinate supplementation was able to restore the normal SDH activity starting from 50µM. Taken globally, these data suggest that P-Et acts as a competitive inhibitor of succinate for SDH activity. In line with these data, automatic docking of P-Et inside the SDH catalytic domain confirmed that P-Et can occupy the succinate binding site in an energetically favorable conformation, mimicking succinate. In conclusion, the reduced activity of mutant ETNK1 leads to the accumulation of new mutations through the reduced competition of P-Et with succinate, increased mitochondrial activity and ROS production. This mechanism can be blocked, at least in vitro, by P-Et supplementation, suppressing the accumulation of new mutations mediated by the ETNK1-dependent mutator phenotype. In vivo studies will address the therapeutic potential of P-Et. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Key Points Whole-exome sequencing reveals the presence of recurrent somatic mutations of ETNK1 in patients with atypical chronic myeloid leukemia. ETNK1 mutations impair the catalytic activity of the enzyme, causing a decrease in the intracellular levels of phosphoethanolamine.

Abstract Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the myelodisplastic-myeloproliferative neoplasms, according to the WHO-2008 classification. From a clinical point of view it closely resembles the classical Chronic Myeloid Leukemia (CML), however it lacks the presence of the Philadelphia chromosome and of the BCR-ABL1 fusion gene. In recent works, we and others characterized the somatic lesions present in the aCML genome, mainly by using Next Generation Sequencing (NGS) technologies, demonstrating the presence of a large set of recurrent somatic mutations involving, among the others, SETBP1, ETNK1, ASXL1, EZH2, CBL, TET2, NRAS and U2AF1 genes. The identification of somatic variants occurring in a large number of genes clearly indicates that the genetic bases of aCML are very heterogeneous, in striking contrast with classical CML. This heterogeneity poses a great challenge to the dissection of the molecular steps required for aCML leukemogenesis. The hierarchical reconstruction of the different mutations occurring in a clonal disorder can have important biological, prognostic and therapeutic repercussions; therefore we started a project focused on the dissection of the aCML clonal evolution steps through the analysis of individual leukemic clones by methylcellulose assays in samples whose mutational status has been previously characterized by matched whole-exome sequencing. Patient CMLPh-019 was characterized by the presence of a complex mutational status, with somatic variants occurring in SETBP1, ETNK1, ASXL1 and CBL genes (Fig. 1a). Targeted resequencing analysis of individual clones revealed the presence of all the 4 variants in 44/60 (73.3%) clones; in 15/60 (25%) we detected the presence of mutated ETNK1, ASXL1 and CBL and wild-type (WT) SETBP1. Of these 15 clones, 33% carried heterozygous and 67% homozygous CBL mutations. In one clone (1.7%) we detected heterozygous ETNK1, homozygous CBL and WT sequences for ASXL1 and SETBP1, suggesting a strong selective pressure towards the acquisition of homozygous CBL mutations. Identification of homozygous CBL mutations in all the main clonal phases suggests that a significant positive selective pressure is associated with this event. Allelic imbalance analysis of CMLPh-019 exome using CEQer revealed that CBL homozygosity is caused by a somatic uniparental disomy event occurring in the telomeric region of the long arm of chromosome 11. Patient CMLPh-005 (Fig. 1b) was mutated in ASXL1, CBL and SETBP1. Targeted analysis done on 68 clones revealed a complex, branching evolution, with 63 clones carrying all the 3 variants. Of them, 47 (74.6%) had a heterozygous and 16 (25.4%) a homozygous CBL variant. Four clones (4.2%) carried ASXL1 and SETBP1 but not CBL mutations, while 1 clone was mutated in ASXL1 and CBL in absence of SETBP1 mutations, which suggests that CBL mutations occurred independently in two different subclones. Also in this case, allelic imbalance analysis of exome data revealed that CBL homozygosity was caused by a telomeric somatic uniparental disomy event. According to exome sequencing, patient CMLPh-003 carried SETBP1 mutation G870S and NRAS variant G12R. Clonal analysis confirmed the presence of SETBP1 G870S in all the clones analyzed, while heterozygous NRAS G12R mutation was detected in 67% (Fig. 1c). Notably in the remaining 33% another heterozygous NRAS variant, G12D, was detected. Retrospective reanalysis of exome data confirmed the presence of the newly identified variant, which had been previously filtered-out from exome data because of the low frequency. Patient CMLPh-013 was mutated in ASXL1, ETNK1, NRAS and SETBP1. Of the 39 clones analyzed, 34 (82.9%) showed the coexistence of ASXL1, ETNK1, NRAS and SETBP1, 4 were mutated in ASXL1, ETNK1 and NRAS and 1 in ETNK1 and NRAS, suggesting that ETNK1 and NRAS were early events, ASXL1 an intermediate one and SETBP1 a late variant (Fig. 1d). Taken globally, these data indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while ASXL1 represents an early/intermediate event and SETBP1 is often a late event. They also suggest that, in the context of aCML, there is a strong selective pressure towards the accumulation of homozygous CBL variants, as already shown in other leukemias. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Disclosures Rea: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.

Abstract Background Der(1;7)(q10;p10) (der(1;7) is an unbalanced translocation recurrently found in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and related disorders. Caused by a recombination between two homologous alphoid sequencing D1Z7 and D7Z1 on chromosomes 1 and 7, respectively, it results in monosomy 7q and trisomy 1q, which is implicated in the pathogenesis of der(1;7)-positive myeloid neoplasms. Previous studies reported frequent co-occurrence of +8 and del(20q), as well as RUNX1 mutations, the genetic and clinical characteristics of this abnormality has not fully been elucidated. Methods In this study, we enrolled a total of 153 cases myeloid neoplasms positive for der(1;7) from Japanese and German cohorts, in which co-occurring genetic lesions were analyzed using whole exome and/or targeted-capture sequencing. An additional 3,223 MDS and related neoplasm cases were also analyzed using targeted-capture sequencing to identify der(1;7)-specific genomic features. Results Ethnicity was evaluated comparing the frequency of der(1;7) in 944 German MDS cases and 763 Japanese MDS cases. Der(1;7) cases were observed at a higher frequency in Japanese MDS cohort compared to German MDS cohort (73/763 cases versus 4/944 cases, p &lt; 0.00001). Der(1;7) cases showed a strong male predominance (86.3%) (p&lt;0.001). Of 153 myeloid neoplasm patients harboring der(1;7), 114 were diagnosed with MDS, 28 with AML, 5 with MDS-MPN and 1 with MPN. Targeted-capture sequencing revealed mutations in common myeloid drivers (n=61) in 96% of der(1;7) cases. The most frequently mutated gene was RUNX1 with 46%, followed by ETNK1 (24.5%) and EZH2 (24.5%). Of interest, ETNK1 mutation was identified as the most unique to der(1;7) when compared to myeloid neoplasm cases without der(1;7) (n=3,066) [odds ratio (OR)=15.06], followed by ETV6 (OR=9.35) and EZH2 (OR=6.52). To further examine the uniqueness of this mutation profile, the mutational profile of der(1;7) was compared to those myeloid neoplasm cases harboring amp(1q) (n=52) and monosomy 7 (n=105). Highly frequent ETV6 and ETNK1 mutations were highly unique to der(1;7) cases when compared to amp(1q) cases (OR=3.72, OR=2.57, respectively). BCOR and ETNK1 mutations were highly unique to der(1;7) cases when compared to monosomy 7 cases (OR=35.88, OR=4.29, respectively). Both amp(1q) and monosomy 7 cases showed a higher mutation rate in TP53 compared to der(1;7) cases (49.1% and 51%, respectively, vs 3.5 %) . From these mutational characteristics, ETNK1 was identified as being the most unique to der(1;7) when compared to amp(1q), monosomy 7 and other myeloid neoplasm cases. ETNK1-mutated der(1;7) cases were featured with eosinophilia (p &lt; 0.0005), a lack of RAS pathway mutations and trisomy 8 when compared to ETNK1-wild type der(1;7) cases. Survival analysis was conducted to elucidate the difference in survival in der(1;7) cases (n=65) versus myeloid neoplasm cases (n=2066). Der(1;7)-harboring myeloid neoplasm cases had a median overall survival of 6.8 months (95% CI, 3.5 to 11.9) and non-der(1;7) harboring myeloid neoplasm cases were 11.8 months (95% CI, 10.5 to 12.6). Thus, der(1;7)-harboring myeloid neoplasm cases had poorer prognosis (p&lt;0.001). Conclusion In conclusion, der(1;7) is an unbalanced translocation that occurs predominantly in males and is seen more frequently in Japanese than Caucasian populations. Der(1;7) cases present with a mutational profile that is distinct from other myeloid neoplasm cases such as those with amp(1q) and monosomy7/del(7q), showing frequency of ETNK1 mutations. Disclosures Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria. Handa: Ono: Honoraria; BMS: Honoraria; Janssen: Honoraria; Daiichi Sankyo: Research Funding; Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding. Ohyashiki: Novartis Pharma: Other: chief clinical trial; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Ogawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

der(1;7)(q10;p10) is a recurrent chromosomal abnormality found in a wide variety of myeloid neoplasms observed in as high as 6% of myelodysplastic syndromes (MDS) in Asian populations, while rarely observed in Caucasian populations. It is thought to be generated by a recombination between two highly homologous centromere alphoid sequences which lead to an unbalanced abnormality of monosomy of 7q and trisomy of 1q. However, despite the presence of -7q, der(1;7) has been associated with a better prognosis compared to monosomy 7 or other del(7q) (-7/del(7q)). In addition to its association with +8 and del(20q), frequent RUNX1 mutations and a paucity of mutated TP53 have been reported in der(1;7) tumors, but otherwise, the molecular features of this abnormality have been poorly characterized in the literature. This is most likely because it is very rare in Caucasians, even though it represents one of the most prevalent lesions among Asian populations. The purpose of our study is to clarify the frequency and mutational landscape of der(1;7) in myeloid neoplasms on the basis of targeted-capture sequencing. A total of 1,707 MDS cases, including 944 German and 763 Japanese cases, were enrolled, from which we identified 73 (4.0%) cases with der(1;7). The prevalence was >20 times higher in Japanese (9.0%) than German (0.43%) cohorts (p<0.0001). We also identified a strong male predominance in der(1;7)-positive cases (90.4%) compared to negative cases. Also including an additional 22 cases, somatic mutations and copy number abnormalities in der(1;7) were interrogated in a total of 95 cases, which included 84 (88.4%) with MDS, 9 (9.5%) with AML, and 2 (2.1%) with MPN. Among MDS patients, 29 were low-risk, 47 were high-risk, and the rest were not specified. In mutation analysis, at least one mutation was detected in 98% of der(1;7) cases, most frequently affecting RUNX1 (42%), followed by EZH2 (26%), and ETNK1 (25%). Copy number analysis showed a high frequency of del(20q) and trisomy 8 in der(1;7) cases: 27.4% and 18.9% respectively. On the basis of mutant cell fractions, most of these mutations were present in subclones acquired within the major population harboring der(1;7). In particular, most of the EZH2 (7q35-q36) mutations were thought to be secondary events in der(1;7)-positive cases, while representing initial events acquired before UPD(7q) or -7/del(7q) in der(1;7)-negative cases. Of interest, der(1;7) was associated with a low frequency of TP53 mutations, which were seen only in 3% of cases with der(1;7), whereas highly prevalent in non-der(1;7) cases with -7/del(7q) (52%), which is concordant with a better clinical outcome was observed in der(1;7) cases compared with non-der(1;7) cases with monosomy 7 or other del(7q). Another unique feature of der(1;7) positive MDS was an extremely high frequency of RUNX1 mutations. However, the most prominent finding with secondary mutations in der(1;7) cases is the frequent hot spot mutation in ETNK1, which were originally reported in 8.8% of myeloid neoplasms with MPN features, like SETBP1 mutations. ENTK1 mutations were found in as many as 25% (23/95) of der(1;7) cases, while rarely seen in -7/del(7q) (1/89) (p<0.0001) or amp(1q) (2/68) (p=0.0001). Despite the high frequency of trisomy 8 observed in der(1;7) cases, none were associated with ETNK1 mutations. In addition, all of the RAS pathway mutations (positive in 16 cases) were observed in der(1;7) cases with wild-type ETNK1, while none were in ETNK1-mutant cases. Morphologically, these ETNK1-mutated der(1;7) cases presented with an increased eosinophil count in peripheral blood (760.9/ul vs. 78.1/ul) (p<0.001), compared to those without EKNK1 mutations, suggesting that ENTK1-mutated der(1;7) cases represent a novel disease entity within der(1;7), characterized by unique genetic features and increased eosinophils. In conclusion, der(1;7) is a genetically and clinically distinct subset of myeloid neoplasms, which showed unique features that are distinct from MDS cases in -7 and other del(7q). Especially, ETNK1 mutations subdivided cases with der(1;7) into two groups of genetically distinct subsets as shown in Figure 1. In the future, inhibition of the kinase activity in ETNK1 could be a novel therapeutic strategy in such a previously unrecognized subset as characterized by der(1;7) and eosinophilia. Figure 1 Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Handa:Ono: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Qiagen Corporation: Patents & Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership.

Our previous findings have shown that the chlorophyllides composites have anticancer activities to breast cancer cell lines (MCF-7 and MDA-MB-231). In the present study, microarray gene expression profiling was utilized to investigate the chlorophyllides anticancer mechanism on the breast cancer cells lines. Results showed that chlorophyllides composites induced upregulation of 43 and 56 differentially expressed genes (DEG) in MCF-7 and MDA-MB-231 cells, respectively. In both cell lines, chlorophyllides composites modulated the expression of annexin A4 (ANXA4), chemokine C-C motif receptor 1 (CCR1), stromal interaction molecule 2 (STIM2), ethanolamine kinase 1 (ETNK1) and member of RAS oncogene family (RAP2B). Further, the KEGG annotation revealed that chlorophyllides composites modulated DEGs that are associated with the endocrine system in MCF-7 cells and with the nervous system in MDA-MB-231 cells, respectively. The expression levels of 9 genes were validated by quantitative reverse transcription PCR (RT-qPCR). The expression of CCR1, STIM2, ETNK1, MAGl1 and TOP2A were upregulated in both chlorophyllides composites treated-MCF-7 and MDA-MB-231 cells. The different expression of NLRC5, SLC7A7 and PKN1 provided valuable information for future investigation and development of novel cancer therapy.

Atypical chronic myeloid leukemia (aCML) is a rare BCR-ABL1 negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in several genes including SETBP1, ASXL1 and ETNK1, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. The clinical prognosis for aCML is poor, with a median overall survival of 18 months after diagnosis, and no established standards of care exist for its treatment. The dissection of the molecular processes underlying aCML leukemogenesis could therefore result decisive in ameliorating the prognosis for aCML. With the aim to provide a comprehensive genomic characterization of aCML and to link the detected alterations with the clinical course of the disease, we applied a high-throughput sequencing strategy to 43 aCML samples, including whole-exome sequencing and RNA sequencing. Our study confirms ASXL1 and SETBP1 as the most frequently mutated genes with a total of 43.2% and 30.2%, respectively; ETNK1 mutations are observed in 14% of patients. An average of 2 mutations per patient was observed [range: 0-5]. We characterized the clonal architecture in a subset of 8 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while SETBP1 mutations represent a late event; interestingly, in the two cases where ASXL1 was mutated together with SETBP1, its mutations occupied an intermediate hierarchical position. CBL mutations, when present, showed a tendency toward reaching homozygosity through somatic uniparental disomy. Stratification based on RNA-sequencing gene expression data (Ramazzotti, Daniele, et al. Nature communications 9.1 (2018): 4453) identified two clearly different populations (26 and 17 patients) in terms of Overall Survival (OS), with 2 year OS of 69.23% [95% IC: 48.21%-86.67%] and 35.29% [95% IC: 14.21%-61.67%] respectively (logrank test for trend: p=0.004, Fig. 1A). In addition, the group with better prognosis showed a higher frequency of ETNK1 mutations (hypergeometric test: p=0.032). We next performed differential gene expression analysis to detect genes differentially expressed between the two patients' populations. This analysis revealed 38 significant genes (t-test p-value adjusted for false discovery rate p<0.01) overexpressed in the group with negative outcome. Notably, the majority of these genes are known cancer drivers, such as IDH2, MEN1, MYC and TP53. Involved pathways include gene transcription and cell differentiation, mitochondrial activity and DNA repair. We then considered RNA-sequencing data for the 4 most significant genes within the previous list (namely DNPH1, GFI1B, PARP1 and POLRMT) to build a classifier capable of associating patients to the respective subtype (better vs. worse prognosis). Our results show that a random forest classifier (Ho, Tin Kam. Proceedings of 3rd international conference on document analysis and recognition. Vol. 1. IEEE, 1995) using the 4 most significant genes achieves a 93.79% accuracy assessed by means of 10 fold cross validation (Fig.1B-C). In conclusion, we present here the first description of a large aCML cohort, in which sequencing data, clonal hierarchy of mutations and gene expression profiles were integrated through bioinformatics analysis. RNA-sequencing data stratification characterizes two groups with different prognosis; a classifier based on the 4 top differently expressed genes accurately predicts patients' outcome. Figure 1. A) Overall Survival curve (Kaplan-Meier curve) at 24 months shows significant different outcomes (p=0.004). B) Random forest classifiers learn multiple decision trees in order to predict outcomes. In the figure, an example of decision tree where nodes are genes and leaves are outcomes (better/worse prognosis). C) Heatmap of expression fold change for the top four differentially expressed genes. Figure 1 Disclosures Rea: BMS: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stagno:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Pfizer: Honoraria, Research Funding; Bristol-Meyers Squibb: Consultancy.

La leucemia mieloide cronica atipica (aCML) è un disordine clonale appartenente al gruppo delle sindromi mielodisplastiche/mieloproliferative (MDS/MPN). Circa il 13% dei casi di aCML è caratterizzato dalla presenza di mutazioni somatiche a carico del gene ETNK1, codificanti per le sostituzioni amminoacidiche H243Y, N244S e G245V. Precedentemente, abbiamo dimostrato che sia in campioni primari di aCML ETNK1-positiva sia nella linea cellulare TF1 trasdotta con ETNK1 mutato, le mutazioni causano una diminuzione dell’attività enzimatica di ETNK1, causando una riduzione nei livelli intracellulari di fosfoetanolamina (P-Et). Per caratterizzare il ruolo funzionale delle mutazioni di ETNK1, ho creato un nuovo modello cellulare isogenico CRISPR/Cas9 nel quale la mutazione N244S è presente come variante in eterozigosi, e ho studiato l’effetto funzionale della modulazione di P-Et applicando diverse strategie, tra cui approcci di metabolimica, lipidomica, analisi di espressione genica, ed esperimenti di respirazione mitocondriale, dimostrando che la mutazione di ETNK1 (i) causa un aumento del potenziale di membrana mitocondriale, (ii) un cambiamento della morfologia mitocondriale, (iii) un aumento della produzione di ROS, (iv) e aumenta il tasso di mutazioni al DNA genomico. Inoltre, ho dimostrato che l’aumentata attività mitocondriale causata dalla mutazione di ETNK1 è dovuta a una diretta competizione tra P-Et e succinato per l’enzima succinato deidrogenasi (complesso II). Infine, esperimenti di ricostruzione della gerarchia clonale delle mutazioni somatiche nei pazienti affetti da aCML indicano che le mutazioni di ETNK1 sono eventi precoci nel processo evolutivo della patologia, suggerendo per ETNK1 un ruolo di induttore di un fenotipo mutante, il quale a sua volta contribuisce all’accumulo di ulteriori mutazioni oncogeniche.
Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the MDS/MPN syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S and G245V substitutions. We previously showed that, in both ETNK1-positive aCML primary samples and TF1 cells transduced with mutated ETNK1, the mutations lead to an impairment of ETNK1 enzymatic activity, responsible for a decrease in the intracellular level of phosphoethanolamine (P-Et). To dissect the functional role of ETNK1 mutations I created a new isogenic CRISPR/Cas9 cellular model in which ETNK1 N244S mutation was present as heterozygous variant, and I investigated the functional effect of P-Et modulation by a combined approach involving metabolomics, lipidomics, whole-transcriptome sequencing, ChIP and mitochondria respiration analyses, showing that it causes (i) increased mitochondria potential, (ii) change in mitochondria morphology, (iii) increased ROS production, (iv) increased gDNA mutation rate. I also showed that the increased mitochondrial activity in presence of ETNK1 mutations is caused by a direct competition between P-Et and succinate for complex II succinate dehydrogenase enzyme. Finally, experiments focused on the reconstruction of the hierarchy of somatic mutations in aCML patients showed that ETNK1 somatic mutations are early events in the subclonal history of aCML, which fits with a role of ETNK1 as an inducer of a mutant phenotype, which in turn would accelerate the accumulation of further oncogenic mutations.