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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.

Differentiation abnormalities are a hallmark of tuberous sclerosis complex (TSC) manifestations; however, the genesis of these abnormalities remains unclear. Here we report on mechanisms controlling the multi-lineage, early neuronal progenitor and neural stem-like cell characteristics of lymphangioleiomyomatosis (LAM) and angiomyolipoma cells. These mechanisms include the activation of a previously unreported Rheb-Notch-Rheb regulatory loop, in which the cyclic binding of Notch1 to the Notch-responsive elements (NREs) on the Rheb promoter is a key event. This binding induces the transactivation of Rheb. The identified NRE2 and NRE3 on the Rheb promoter are important to Notch-dependent promoter activity. Notch cooperates with Rheb to block cell differentiation via similar mechanisms in mouse models of TSC. Cell-specific loss of Tsc1 within nestin-expressing cells in adult mice leads to the formation of kidney cysts, renal intraepithelial neoplasia, and invasive papillary renal carcinoma., Tuberous sclerosis complex (TSC) is a rare genetic condition causing tumours with differentiation abnormalities; however the molecular mechanisms causing these defects are unclear. Here the authors show that Notch cooperates with Rheb to block cell differentiation forming a regulatory loop that could underlie TSC tumorigenesis.

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.

Abstract 2947 It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation. In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model. Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction. Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 1811 Symptomatic multiple myeloma (MM), smoldering MM (SMM) and monoclonal gammopathy of uncertain significance (MGUS) are well known different pathological and clinical entities of plasma cell (PC) disorders. Nevertheless molecular studies performed on clonal CD138+ PC do not clear distinguished these disorders that share common alterations. Studies focusing on the presence of potential molecular alterations in the microenvironment cells are ongoing. Because monocytes are the cells primarily involved in osteoclastogenesis, angiogenesis and immune system disfuction, that are the hallmark of symptomatic MM compared to SMM and MGUS, in this study we have analyzed the transcriptional profile of the bone marrow (BM) CD14+ cells in these settings of patients. BM CD14+ monocytes were purified from a total cohort of 36 patients with PC disorders including 21 patients with symptomatic MM, 8 patients with SMM and 7 patients with MGUS. CD14+ cells were isolated from the CD138 negative fraction of BM samples of patients by immunomagnetic method with anti-CD14 monoclonal antibody conjugated with microbeads. The presence of potential haemopoietic and CD138+ contaminating cells was excluded by FACS analysis. Only samples with CD14 purity greater than 95% were analyzed by microarrays by GeneChip® HG-U133Plus 2.0 arrays (Affymetrix®) (13 MM, 8 SMM and 7 MGUS). Data obtained were then validated on selected genes by Real-Time quantitative PCR. A multiclass analysis identified 14 differentially expressed genes, which characterized MGUS vs SMM vs symptomatic MM. A supervised analysis between symptomatic MM vs. SMM and MGUS samples identified 101 genes differentially expressed in CD14+ (58 genes up-regulated in MM vs SMM and MGUS and 43 genes donwregulated). Interestingly, among the differentially expressed genes we found that cytokines and cytokine receptors (IL21, IL21R, IL15, IL15R), chemokines (CXCL10, CXCL11) and interferon-inducible proteins (IFI27, IFI44) were up-regulated in CD14+ of MM patients as compared to SMM and MGUS. A supervised analysis between MM and MGUS identified 6 differentially expressed genes in CD14+ whereas 37 genes distinguished MM and SMM patients. Notably the SLAMF7 (CS1) gene recently indentified as a therapeutic target in CD138+ MM cells was up-regulated in CD14+ monocytes of MM patients as compared either to MGUS alone or to MGUS plus SMM could be a potential candidate gene. Overall our preliminary results indicate that a different transcriptional fingerprint may be identified in BM CD14+ cells of patients with symptomatic MM as compared to those with indolent PC disorders such as SMM and MGUS with a greater number of differentially expressed genes between symptomatic MM and SMM patient rather than between MM and MGUS. Disclosures: No relevant conflicts of interest to declare.