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PDF - K, Supplementary Figure 5. Interference of NUP98-fusions with APC/C activity involves primarily CDC20. HEK293 cells were transfected with a control siRNA or with siRNAs against the indicated proteins (CDC20 or CDH1), together with the expression constructs for NUP98 or NUP98-fusion proteins (indicated on the left). Immunoblot analysis shows Securin (SEC) or CyclinB1 (CCNB1) levels after Nocodazole treatment for the indicated time points. Anti-betaactin (betaACT) was used as a loading control.

PDF - 2287K, Supplementary Figure 3. Mitotic index in transfected and in nocodazole arrested HEK293 cells. A, Mitotic index of control untransfected, nocodazole-arrested HEK293 and U937 cells. Cells were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry. B, Immunoblot analysis of the expression of the indicated proteins in HEK293 cells transiently transfected with expression constructs for NUP98, NUP98-HOXD13, NUP98-LOC, NUP98-HHEX, treated with nocodazole, and harvested at the indicated time points. An arrowhead indicates the NUP98-HOXD13 band just below that of endogenous NUP98. CyclinB1 (CCNB1), Geminin (GMN) and securin (SEC) amounts were detected C, HEK293 cells, transiently transfected with expression constructs for the indicated proteins, were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry.

PDF - 1659K, Supplementary Figure 4. siRNA knock-down experiments. A, Immunoblots showing the effects of siRNAs on CDC20 or CDH1 protein expression. Anti-betaactin was used as a loading control. Graphs representing immunoblot quantitations are shown on the right side. B, Cell cycle analysis using propidium iodide staining and flow citometry on control siRNA-treated HEK293 cells (left side), and on siRNA-treated HEK293 cells expressing the NUP98-HOXD13 oncogene (right side).

PDF - 77K, Legends to Supplementary Figures

PDF - 2289K, Supplemental Figure 1. Expression of NUP98 and of NUP98 fusion oncoproteins in HEK293 cells. A, Schematic representation of the structures of human NUP98 (AAC50366) and of its chimeric oncoproteins. The GLFG repeats and the Rae1-interacting GLEBS domain are shown as black and dark grey boxes, respectively. The N-terminal portion of NUP98 is similar in the studied chimeras, spanning aa 1 to aa 469 in NUP98-HOXD13, and aa 1 to aa 514 in NLOC and NHHEX fusions. The HOXD13 (aa 252 to 335, AAC51635), LOC348801 isoform1 (aa 1 to 178, AAI26363), and HHEX (aa 140 to 270, NP_002720) C-terminal moieties are shown as coloured boxes. The homeodomain regions (HD) of HOXD13 and HHEX are indicated as a grey box within the coloured one. Also shown is the structure of NUP98-HOXD13IQN, which carries mutations of three amino acids within the homeodomain (white), impairing DNA binding. B, Western blot analysis showing the expression levels of the indicated proteins transiently expressed in HEK293 cells (T0), and after Nocodazole treatment at the indicated time points. Immunoblottings were performed with anti-NUP98 and anti-betaactin antibodies as a loading control.

PDF - 1450K, Supplementary Figure 2. Proteasome inhibition rescues NUP98-fusions effects on Securin and Cyclin B degradation. Western blot analysis showing securin (SEC) and cyclin B (CCNB1) levels in HEK293 cells transfected with the indicated constructs after treatment with Nocodazole or Nocodazole and the proteasome inhibitor MG132 for 24 hours. An increase of CCNB1 levels can be appreciated in control cells following MG132 treatment as the result of efficient proteasome inactivation. Anti-betaactin antibody (betaACT) was used as a loading control.

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)Cdc20 and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability. Cancer Res; 74(4); 1079–90. ©2013 AACR.

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Simple Summary Myelofibrosis (MF) displays the worst prognosis among Philadelphia-negative chronic myeloproliferative neoplasms. There is no curative therapy for MF, except for bone marrow transplantation, which however has a consistent percentage of failure. There is thus an urgent need of novel biomarkers to complement current stratification models and to enable better management of patients. To address this issue, we herein measured the plasma levels of several long noncoding RNAs (lncRNAs). Circulating lncRNAs has been already largely described as potential non-invasive biomarkers in cancers. In our study we unveiled that LINC01268, MALAT1 (both p < 0.0001) and GAS5 (p = 0.0003) plasma levels are significantly higher in MF patients if compared with healthy donors, and their increased plasma levels correlate with several detrimental features in MF. Among them, LINC01268 is an independent variable for both OS (p = 0.0297) and LFS (p = 0.0479), thus representing a putative new biomarker suitable for integrate contemporary prognostic models. Abstract Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients’ cohort, it could be used for further studies to design an updated classification model for MF patients.

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decision, we studied single cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid Leukemia (AML) while receiving Ruxolitinib. Single cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in chronic phase. Single cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

// Sebastian Fantini 1 , Valentina Salsi 1, 2 , Luca Reggiani 3 , Antonino Maiorana 3 , Vincenzo Zappavigna 1 1 Department of Life Sciences, Modena 41125, Italy 2 Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto, University of Modena and Reggio Emilia, Modena 41125, Italy 3 Dipartimento di Medicina Diagnostica, Clinica e di Sanita Pubblica, University of Modena and Reggio Emilia, Modena 41124, Italy Correspondence to: Vincenzo Zappavigna, email: zappavigna.vincenzo@unimore.it Keywords: colorectal cancer, gene regulation, gene expression, molecular mechanisms, molecular carcinogenesis Received: April 18, 2016 Accepted: November 11, 2016 Published: November 25, 2016 ABSTRACT Objective: To explore the possible misexpression of the microRNA miR-196b in colorectal cancer (CRC) and its role in controlling the expression of GATA6, a putative target gene crucial to intestinal cell homeostasis and tumorigenesis. Design: The expression of miR-196b was analysed by qRT-PCR in surgical resection samples from a cohort of sporadic colon cancer patients. Manipulations of miR-196b expression were performed to demonstrate its inhibition of GATA6 protein levels. Results: We found that miR-196b is significantly upregulated in pre-treatment surgical resection samples from a cohort of sporadic colon cancer patients. The upregulation of miR-196b correlates with less severe clinicopathological characteristics, such as early tumor stage and absence of lymph node metastases. We show that in CRC cells, miR-196b targets the mRNA of GATA6, a transcription factor involved in the homeostasis and differentiation of intestinal epithelial cells, and a positive regulator of the Wnt/β-catenin pathway. We moreover found that the increase of miR-196b correlates with a reduced GATA6 protein expression in colon cancer patients. Conclusion: Our results establish miR-196b as a post-transcriptional inhibitor of GATA6 in CRC cells, implicating miR-196b function in gene regulatory pathways crucial to intestinal cell homeostasis and tumorigenesis. Our results furthermore suggest a role of miR-196b expression in CRC, as an antagonist of GATA6 function in tumor cells, thus providing the basis for a potential targeting strategy for the treatment of CRC.

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34

The miR-196 miRNA gene family located within the Hox gene clusters has been shown to function during embryogenesis and to be aberrantly expressed in various malignancies, including leukaemia, melanoma, and colorectal cancer. Despite its involvement in numerous biological processes , the control of miR-196 expression is still poorly defined. We identified the miR-196b promoter and found that the mature miR-196b originates from a large, non-coding primary transcript , which starts within an autonomous TATA box promoter and is not in physical continuity with either the Hoxa10 or Hoxa9 main primary transcripts. A ~ 680 bp genomic fragment , spanning the pri-miR-196b transcription start site , is sufficient to recapitulate the neural tube expression pattern of miR-196 during embryogenesis. This region contains potential binding sites for Cdx and 5′Hox transcription factors. Two of these sites revealed to be necessary for neural tube expression and were bound in vivo by Cdx2 and Hoxd13. We show that Cdx2 is required for miR-196 expression and that both Cdx2 and 5′Hox, but not 3′Hox, are able to activate the miR-196b promoter. The possible role of Cdx2- and 5′Hox-mediated regulation of miR-196 expression in vertebrate anterior–posterior (AP) axis formation during embryogenesis is discussed.

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing μ-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores μ-protocadherin expression and promotes the sequestration of β-catenin to the plasma membrane. Here, we show that 5-ASA–induced μ-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated β-catenin inhibition is caused by μ-protocadherin–dependent sequestration of β-catenin to the plasma membrane and by the direct binding of KLF4 to β-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA. Cancer Prev Res; 11(8); 503–10. ©2018 AACR.

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)Cdc20 and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability. Cancer Res; 74(4); 1079–90. ©2013 AACR.

Genes expressed in the somatopleuric mesoderm, the embryonic domain giving rise to the vertebrate pelvis, appear important for pelvic girdle formation. Among such genes, Pbx family members and Emx2 were found to genetically interact in hindlimb and pectoral girdle formation. Here, we generated compound mutant embryos carrying combinations of mutated alleles for Pbx1, Pbx2, and Pbx3, as well as Pbx1 and Emx2, to examine potential genetic interactions during pelvic development. Indeed, Pbx genes share overlapping functions and Pbx1 and Emx2 genetically interact in pelvic formation. We show that, in compound Pbx1;Pbx2 and Pbx1;Emx2 mutants, pelvic mesenchymal condensation is markedly perturbed, indicative of an upstream control by these homeoproteins. We establish that expression of Tbx15, Prrx1, and Pax1, among other genes involved in the specification and development of select pelvic structures, is altered in our compound mutants. Lastly, we identify potential Pbx1-Emx2–regulated enhancers for Tbx15, Prrx1, and Pax1, using bioinformatics analyses. Developmental Dynamics 240:1173–1189, 2011. © 2011 Wiley-Liss, Inc.

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

The 5' members of the HoxD gene cluster (paralogous groups 9-13) are crucial for correct vertebrate limb patterning. Mutations in the HOXD13 gene have been found to cause synpolydactyly (SPD) and other limb malformations in human. We report the identification in a Greek family of a variant form of SPD caused by a novel missense mutation that substitutes glycine for valine in position 220 (G220V) of HOXD13. This mutation represents the first substitution of an amino acid located outside of the HOXD13 homeodomain that causes autopodal limb malformations. We have characterized this mutation at the molecular level and found that the G220V substitution causes a significant impairment of the capacity of HOXD13 to bind DNA and regulate transcription. HOXD13(G220V) was found to be deficient in both activating and repressing transcription through HOXD13-responsive regulatory elements. In accordance with these results, a comparison of the activities of HOXD13 and HOXD13(G220V) in vivo, using retrovirus-mediated misexpression in developing chick limbs, showed that the G220V mutation impairs the capacity of HOXD13 to perturb the development of proximal limb skeletal elements and to ectopically activate the transcription of the Hand2 target gene. We moreover show that the G220V mutation compromises the stability of the HOXD13 protein within cells and causes its partial accumulation in the cytosol in the form of subtle aggregates. Taken together, our results establish that the G220V substitution does not produce a dominant-negative effect or a gain-of-function, but represents a dominant loss-of-function mutation revealing haploinsufficiency of HOXD13 in human.

The influence of the Zn(2+) concentration and temperature on the electrochemical reduction of O(2) in a solution of zinc bis(trifluoromethanesulfonyl)imide (Zn(TFSI)(2)) salt in 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (PYR(14)TFSI) ionic liquid is presented. ZnO nanocrystalline films were then electrodeposited, under enhanced O(2) reduction, at temperatures in the 75-150 °C range. Their morphology, chemical composition, structural and optical properties were analyzed. In contrast to the polar-oriented ZnO usually obtained from aqueous and conventional solvent based electrolytes, nanocrystalline films oriented along non-polar directions, (11 ̅10) and (11 ̅20), were obtained from this ionic liquid electrolyte. A significant content of carbon was detected in the films, pointing to the active participation and crucial effect of pyrrolidinium cation (and/or byproducts) during the electrodeposition. The films showed semiconducting behavior with an optical gap between 3.43 and 3.53 eV as measured by optical transmittance. Their room temperature photoluminescence spectra exhibited two different bands centered at ∼3.4 and ∼2.2 eV. The intensity ratio between both bands was found to depend on the deposition temperature. This work demonstrates the great potential of ionic liquids based electrolytes for the electrodeposition of ZnO nanocrystalline thin films with innovative microstructural and optoelectronic properties.