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Supplementary Data from Effects of Src kinase inhibition induced by dasatinib in non–small cell lung cancer cell lines treated with cisplatin

Supplementary Figure and Table Legend from Effects of Src kinase inhibition induced by dasatinib in non–small cell lung cancer cell lines treated with cisplatin

Supplementary Figure 1 from Effects of Src kinase inhibition induced by dasatinib in non–small cell lung cancer cell lines treated with cisplatin

Supplementary Table 1 from Effects of Src kinase inhibition induced by dasatinib in non–small cell lung cancer cell lines treated with cisplatin

c-Src is a tyrosine kinase involved in tumor proliferation, migration, and angiogenesis and has been shown to modulate the cytotoxicity following cisplatin-induced DNA damages. c-Src is frequently activated in non–small cell lung cancer (NSCLC) tissues and cell lines, but no preclinical data regarding the effects of the novel potent Src inhibitor, dasatinib (BMS-354825), in the modulation of cisplatin resistance are currently available. The present study reports that treatment with dasatinib completely abrogated Src phosphorylation in the majority of the NSCLC cell lines tested (n = 7), with modest effects on cell proliferation and survival. In five cell lines, a higher cytotoxicity was observed delivering cisplatin in combination with dasatinib: the most evident effects were found in the squamous H520 cells due to the effective block of cisplatin-induced Src phosphorylation. Moreover, dasatinib treatment significantly blocked cisplatin-induced transcription of a panel of DNA repair and synthesis genes. In addition, a real-time PCR analysis done on tumor and matched normal lung specimens from 44 surgically resected NSCLC patients showed that Src transcripts are significantly upregulated in 23% of cases. In conclusion, Src-directed therapeutic strategies could interfere with cisplatin resistance, possibly allowing to reduce cisplatin doses, thus improving its efficacy. The data of this study support further clinical studies aimed to evaluate the efficacy of Src-inhibiting agents in combination with cisplatin in the treatment of NSCLC. [Mol Cancer Ther 2009;8(11):3066–74]

Hypoxia is a critical condition that governs survival, self-renewal, quiescence, metabolic shift and refractoriness to leukemic stem cell (LSC) therapy. The present study aims to investigate the hypoxia-driven regulation of the mammalian Target of the Rapamycin-2 (mTORC2) complex to unravel it as a novel potential target in chronic myeloid leukemia (CML) therapeutic strategies. After inducing hypoxia in a CML cell line model, we investigated the activities of mTORC1 and mTORC2. Surprisingly, we detected a significant activation of mTORC2 at the expense of mTORC1, accompanied by the nuclear localization of the main substrate phospho-Akt (Ser473). Moreover, the Gene Ontology analysis of CML patients’ CD34+ cells showed enrichment in the mTORC2 signature, further strengthening our data. The deregulation of mTOR complexes highlights how hypoxia could be crucial in CML development. In conclusion, we propose a mechanism by which CML cells residing under a low-oxygen tension, i.e., in the leukemia quiescent LSCs, singularly regulate the mTORC2 and its downstream effectors.

Large HERC E3 ubiquitin ligase family members, HERC1 and HERC2, are staggeringly complex proteins that can intervene in a wide range of biological processes, such as cell proliferation, DNA repair, neurodevelopment, and inflammation. Therefore, mutations or dysregulation of large HERCs is associated with neurological disorders, DNA repair defects, and cancer. Though their role in solid tumors started to be investigated some years ago, our knowledge about HERCs in non-solid neoplasm is greatly lagging behind. Chronic Myeloid Leukemia (CML) is a model onco-hematological disorder because of its unique and unambiguous relation between genotype and phenotype due to a single genetic alteration. In the present study, we ascertained that the presence of the BCR-ABL fusion gene was inversely associated with the expression of the HERC1 and HERC2 genes. Upon the achievement of remission, both HERC1 and HERC2 mRNAs raised again to levels comparable to those of the healthy donors. Additionally, our survey unveiled that their gene expression is sensitive to different Tyrosine Kinases Inhibitors (TKIs) in a time-dependent fashion. Interestingly, for the first time, we also observed a differential HERC1 expression when the leukemic cell lines were induced to differentiate towards different lineages revealing that HERC1 protein expression is associated with the differentiation process in a lineage-specific manner. Taken together, our findings suggest that HERC1 might act as a novel potential player in blood cell differentiation. Overall, we believe that our results are beneficial to initiate exploring the role/s of large HERCs in non-solid neoplasms.

Large HERC E3 ubiquitin ligase family members, HERC1 and HERC2, are staggeringly complex proteins that can intervene in a wide range of biological processes, such as cell proliferation, DNA repair, neurodevelopment, and inflammation. Therefore, mutations or dysregulation of large HERCs is associated with neurological disorders, DNA repair defects, and cancer. Though their role in solid tumors started to be investigated some years ago, our knowledge about HERCs in non-solid neoplasm is greatly lagging behind. Chronic Myeloid Leukemia (CML) is a model onco-hematological disorder because of its unique and unambiguous relation between genotype and phenotype due to a single genetic alteration. In the present study, we ascertained that the presence of the BCR-ABL fusion gene was inversely associated with the expression of the HERC1 and HERC2 genes. Upon the achievement of remission, both HERC1 and HERC2 mRNAs raised again to levels comparable to those of the healthy donors. Additionally, our survey unveiled that their gene expression is sensitive to different Tyrosine Kinases Inhibitors (TKIs) in a time-dependent fashion. Interestingly, for the first time, we also observed a differential HERC1 expression when the leukemic cell lines were induced to differentiate towards different lineages revealing that HERC1 protein expression is associated with the differentiation process in a lineage-specific manner. Taken together, our findings suggest that HERC1 might act as a novel potential player in blood cell differentiation. Overall, we believe that our results are beneficial to initiate exploring the role/s of large HERCs in non-solid neoplasms.

In aerobic organisms, oxygen is essential for efficient energy production, and it acts as the last acceptor of the mitochondrial electron transport chain and as regulator of gene expression. However, excessive oxygen can lead to production of deleterious reactive oxygen species. Therefore, the directed migration of single cells or cell clumps from hypoxic areas toward a region of optimal oxygen concentration, named aerotaxis, can be considered an adaptive mechanism that plays a major role in biological and pathological processes. One relevant example is the development of O2 gradients when tumors grow beyond their vascular supply, leading frequently to metastasis. In higher eukaryotic organisms, aerotaxis has only recently begun to be explored, but genetically amenable model organisms suitable to dissect this process remain an unmet need. In this regard, we sought to assess whether Dictyostelium cells, which are an established model for chemotaxis and other motility processes, could sense oxygen gradients and move directionally in their response. By assessing different physical parameters, our findings indicate that both growing and starving Dictyostelium cells under hypoxic conditions migrate directionally toward regions of higher O2 concentration. This migration is characterized by a specific pattern of cell arrangement. A thickened circular front of high cell density (corona) forms in the cell cluster and persistently moves following the oxygen gradient. Cells in the colony center, where hypoxia is more severe, are less motile and display a rounded shape. Aggregation-competent cells forming streams by chemotaxis, when confined under hypoxic conditions, undergo stream or aggregate fragmentation, giving rise to multiple small loose aggregates that coordinately move toward regions of higher O2 concentration. By testing a panel of mutants defective in chemotactic signaling, and a catalase-deficient strain, we found that the latter and the pkbR1null exhibited altered migration patterns. Our results suggest that in Dictyostelium, like in mammalian cells, an intracellular accumulation of hydrogen peroxide favors the migration toward optimal oxygen concentration. Furthermore, differently from chemotaxis, this oxygen-driven migration is a G protein-independent process.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.

Simple Summary The pathological role/s of the HERC family members has recently been initiated to be explored in few solid tumors and the assessment of their transcript amount reveals that they might act as effective prognostic factors. However, evidence concerning the non-solid tumors, and especially myeloid related neoplasms, is currently lacking. In the present article for the first time we provide original data for a clear and well-defined association between the gene expression level of a giant HERC E3 ubiquitin ligase family member, HERC1, and some myeloid related disorders, namely Acute Myeloid Leukemia, Myeloproliferative neoplasms and Chronic Myeloid Leukemia. Furthermore, our findings unveil that the HERC1 protein physically interacts, likely forming a very large supramolecular complex, and it is a putative BCR-ABL1 tyrosine kinase substrate. We hope that this work will contribute to the advance of our understanding of the roles played by the giant HERCs in myeloid related neoplasms. Abstract HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that HERC1 gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm HERC1 level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells HERC1 mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of HERC1 in myeloid related blood disorders.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9, 22)(q34, q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

HECT ubiquitin ligases are key components of the eukaryotic ubiquitin-proteasome system controlling different cellular physiological aspects as well as the genesis of several human diseases. Among the HECT family, the HERC subfamily members are characterized by having one or more RCC1-like domains, a C-terminal HECT domain and the molecular mass ranging approximately from 120 kDa to 500 kDa. Due to their large size, some of them are refractory to functional characterization. We have recently identified and functionally characterized a novel large HECT member in Dictyostelium discoideum that, in many aspects, exhibits structural similarities with the mammalian large HERC1. In the present minireview, we shortly summarize and revise the current phylogenetic history of HERC proteins among the different living organisms.

Nandrolone decanoate (ND), an anabolic-androgenic steroid prohibited in collegiate and professional sports, is associated with detrimental cardiovascular effects through redox-dependent mechanisms. We previously observed that high-dose short-term ND administration (15 mg/kg for 2 weeks) did not induce left heart ventricular hypertrophy and, paradoxically, improved postischemic response, whereas chronic ND treatment (5 mg/kg twice a week for 10 weeks) significantly reduced the cardioprotective effect of postconditioning, with an increase in infarct size and a decrease in cardiac performance. We wanted to determine whether short-term ND administration could affect the oxidative redox status in animals exposed to acute restraint stress. Our hypothesis was that, depending on treatment schedule, ND may have a double-edged sword effect. Measurement of malondialdehyde and 4-hydroxynonenal, two oxidative stress markers, in rat plasma and left heart ventricular tissue, revealed that the levels of both markers were increased in animals exposed to restraint stress, whereas no increase in marker levels was noted in animals pretreated with ND, indicating a possible protective action of ND against stress-induced oxidative damage. Furthermore, isolation and identification of proteins extracted from the left heart ventricular tissue samples of rats pretreated or not with ND and exposed to acute stress showed a prevalent expression of enzymes involved in amino acid synthesis and energy metabolism. Among other proteins, peroxiredoxin 6 and alpha B-crystallin, both involved in the oxidative stress response, were predominantly expressed in the left heart ventricular tissues of the ND-pretreated rats. In conclusion, ND seems to reduce oxidative stress by inducing the expression of antioxidant proteins in the hearts of restraint-stressed animals, thus contributing to amelioration of postischemic heart performance.

By protein quality control and degradation, the ubiquitin system drives many essential regulatory processes such as cell cycle and division, signalling, DNA replication and repair. Therefore, dysfunctions in the ubiquitin system lead to many human disease states. However, despite the immense progress made over the last couple of decades, it appears that the ubiquitin system is more complex and multi-faced than formerly expected. In addition to a rich repertoire of ubiquitin, ubiquitin conjugating and de-ubiquitylating enzymes, the social amoeba Dictyostelium discoideum genome encodes also for a wide array of ubiquitin binding domain-containing proteins, thus offering the possibility to explore the biology of the ubiquitin system from cell and molecular biology points of view. We here provide an overview on the current knowledge about the Ub-system components and we discuss how Dictyostelium might be an outstanding eukaryotic cell model for unravelling the still mostly unknown ubiquitination mechanisms of some human diseases.

Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the

Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the Dictyostelium piaA gene and its human ortholog, RICTOR. The two conserved residues are spaced ∼50 amino acids apart, and both are embedded within a conserved region falling in between the Ras-GEFN2 and Rictor­_V domains. The effects of point mutations on the mTORC2 activity and integrity were assessed by biochemical and functional assays. In both cases, these equivalent point mutations in the mammalian RICTOR and DictyosteliumpiaA gene impaired mTORC2 activity and integrity. Our data indicate that the two glycine residues are essential for the maintenance of mTORC2 activity and integrity in organisms that appear to be distantly related, suggesting that they have a evolutionarily conserved role in the assembly and proper mTORC2 functioning.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.

// Valentina Rosso 1 , Jessica Petiti 1 , Enrico Bracco 2 , Roberto Pedrola 1 , Francesca Carnuccio 1 , Elisabetta Signorino 1 , Sonia Carturan 1 , Chiara Calabrese 1 , Giada Bot-Sartor 1 , Michela Ronconi 1 , Anna Serra 1 , Giuseppe Saglio 1 , Francesco Frassoni 3 , Daniela Cilloni 1 1 Department of Clinical and Biological Sciences, University of Turin, Turin, Italy 2 Department of Oncology, University of Turin, Turin, Italy 3 Department of Pediatric Hemato-Oncology and Stem Cell, Cellular Therapy Laboratory, Institute G. Gaslini, Genova, Italy Correspondence to: Daniela Cilloni, email: daniela.cilloni@unito.it Keywords: CALR, PNA, MPN, PCR clamping, diagnostic assay Received: September 14, 2016 Accepted: December 15, 2016 Published: December 23, 2016 ABSTRACT The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene ( CALR ) were detected in 56–88% of JAK2/MPL -negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs. We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.

// Valentina Monica 1 , Marco Lo Iacono 1 , Enrico Bracco 1 , Simone Busso 1 , Laura Di Blasio 2 , Luca Primo 2 , Barbara Peracino 3 , Mauro Papotti 1 , Giorgio Scagliotti 1 1 Department of Oncology, University of Torino, San Luigi Hospital, Orbassano, Torino, Italy 2 Department of Oncology, University of Torino, IRCCS Candiolo, Torino, Italy 3 Department of Clinical and Biological Sciences, University of Torino, San Luigi Hospital, Orbassano, Torino, Italy Correspondence to: Valentina Monica, email: valentina.monica@unito.it Keywords: pemetrexed, dasatinib, malignant pleural mesothelioma, cell lines, thymidylate synthase Received: August 17, 2015 Accepted: June 16, 2016 Published: July 06, 2016 ABSTRACT Background: Thymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated. Results: MPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling. Cell lines and Methods: In three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed. Conclusions: These data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.

Acute myeloid leukemia is mainly characterized by a complex and dynamic genomic instability. Next-generation sequencing has significantly improved the ability of diagnostic research to molecularly characterize and stratify patients. This detailed outcome allowed the discovery of new therapeutic targets and predictive biomarkers, which led to develop novel compounds (e.g., IDH 1 and 2 inhibitors), nowadays commonly used for the treatment of adult relapsed or refractory AML. In this review we summarize the most relevant mutations affecting tumor suppressor genes that contribute to the onset and progression of AML pathology. Epigenetic modifications (TET2, IDH1 and IDH2, DNMT3A, ASXL1, WT1, EZH2), DNA repair dysregulation (TP53, NPM1), cell cycle inhibition and deficiency in differentiation (NPM1, CEBPA, TP53 and GATA2) as a consequence of somatic mutations come out as key elements in acute myeloid leukemia and may contribute to relapse and resistance to therapies. Moreover, spliceosomal machinery mutations identified in the last years, even if in a small cohort of acute myeloid leukemia patients, suggested a new opportunity to exploit therapeutically. Targeting these cellular markers will be the main challenge in the near future in an attempt to eradicate leukemia stem cells.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

Iron, zinc, and copper play fundamental roles in eucaryotes and procaryotes, and their bioavailability regulates host-pathogen interactions. For intracellular pathogens, the source of metals is the cytoplasm of the host, which in turn manipulates intracellular metal traffic following pathogen recognition. It is established that iron is withheld from the pathogen-containing vacuole, whereas for copper and zinc the evidence is unclear. Most infection studies in mammals have concentrated on effects of metal deficiency/overloading at organismal level. Thus, zinc deficiency or supplementation correlate with high risk of respiratory tract infection or recovery from severe infection, respectively. Iron, zinc, and copper deficiency or overload affects lymphocyte proliferation/maturation, and thus the adaptive immune response. Whether they regulate innate immunity at macrophage level is open, except for iron. The early identification in a mouse mutant susceptible to mycobacterial infection of the iron transporter Nramp1 allowed dissecting Nramp1 role in phagocytes, from the social amoeba Dictyostelium to macrophages. Nramp1 regulates iron efflux from the phagosomes, thus starving pathogenic bacteria for iron. Similar studies for zinc or copper are scant, due to the large number of copper and zinc transporters. In Dictyostelium, zinc and copper transporters include 11 and 6 members, respectively. To assess the role of zinc or copper in Dictyostelium, cells were grown under conditions of metal depletion or excess and tested for resistance to Legionella pneumophila infection. Iron shortage or overload inhibited Dictyostelium cell growth within few generations. Surprisingly, zinc or copper depletion failed to affect growth. Zinc or copper overloading inhibited cell growth at, respectively, 50- or 500-fold the physiological concentration, suggesting very efficient control of their homeostasis, as confirmed by Inductively Coupled Plasma Mass Spectrometry quantification of cellular metals. Legionella infection was inhibited or enhanced in cells grown under iron shortage or overload, respectively, confirming a major role for iron in controlling resistance to pathogens. In contrast, zinc and copper depletion or excess during growth did not affect Legionella infection. Using Zinpyr-1 as fluorescent sensor, we show that zinc accumulates in endo-lysosomal vesicles, including phagosomes, and the contractile vacuole. Furthermore, we provide evidence for permeabilization of the Legionella-containing vacuole during bacterial proliferation.

Signal transduction through G-protein-coupled receptors (GPCRs) is central for the regulation of virtually all cellular functions, and it has been widely implicated in human diseases. These receptors activate a common molecular switch that is represented by the heterotrimeric G-protein generating a number of second messengers (cAMP, cGMP, DAG, IP3, Ca2+ etc.), leading to a plethora of diverse cellular responses. Spatiotemporal regulation of signals generated by a given GPCR is crucial for proper signalling and is accomplished by a series of biochemical modifications. Over the past few years, it has become evident that many signalling proteins also undergo ubiquitination, a posttranslational modification that typically leads to protein degradation, but also mediates processes such as protein-protein interaction and protein subcellular localization. The social amoeba Dictyostelium discoideum has proven to be an excellent model to investigate signal transduction triggered by GPCR activation, as cAMP signalling via GPCR is a major regulator of chemotaxis, cell differentiation, and multicellular morphogenesis. Ubiquitin ligases have been recently involved in these processes. In the present review, we will summarize the most significant pathways activated upon GPCRs stimulation and discuss the role played by ubiquitination in Dictyostelium cells.

Cyclic AMP (cAMP) binding to G-protein-coupled receptors (GPCRs) orchestrates chemotaxis and development in Dictyostelium. By activating the RasC–TORC2–PKB (PKB is also known as AKT in mammals) module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia subunit (also known as Rictor in mammals) are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia mutant by random gene disruption led to selection of two suppressor mutants in which spontaneous chemotaxis and development were restored. PKB phosphorylation and chemotactic cell polarization were rescued, whereas Pia-dependent ACA stimulation was not restored but bypassed, leading to cAMP-dependent developmental gene expression. Knocking out the gene encoding the adenylylcyclase B (ACB) in the parental strain showed ACB to be essential for this process. The gene tagged in the suppressor mutants encodes a newly unidentified HECT ubiquitin ligase that is homologous to mammalian HERC1, but harbours a pleckstrin homology domain. Expression of the isolated wild-type HECT domain, but not a mutant HECT C5185S form, from this protein was sufficient to reconstitute the parental phenotype. The new ubiquitin ligase appears to regulate cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation.

ATF2 is a transcription factor involved in stress and DNA damage. A correlation between ATF2 JNK-mediated activation and resistance to damaging agents has already been reported. The purpose of the present study was to investigate whether ATF2 may have a role in acquired resistance to cisplatin in non-small cell lung cancer (NSCLC). mRNA and protein analysis on matched cancer and corresponding normal tissues from surgically resected NSCLC have been performed. Furthermore, in NSCLC cell lines, ATF2 expression levels were evaluated and correlated to platinum (CDDP) resistance. Celastrol-mediated ATF2/cJUN activity was measured. High expression levels of both ATF2 transcript and proteins were observed in lung cancer specimens (p << 0.01, Log2 (FC) = +4.7). CDDP-resistant NSCLC cell lines expressed high levels of ATF2 protein. By contrast, Celastrol-mediated ATF2/cJUN functional inhibition restored the response to CDDP. Moreover, ATF2 protein activation correlates with worse outcome in advanced CDDP-treated patients. For the first time, it has been shown NSCLC ATF2 upregulation at both mRNA/protein levels in NSCLC. In addition, we reported that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Altogether, our results indicate a potential increase in CDDP sensitivity, on Celastrol-mediated ATF2/cJUN inhibition. These data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.

Background: The TP73 and TP63 genes are members of the p53 tumor suppressor family and are expressed in different N -terminal isoforms either with proapoptotic (transactivation domain, TA) and antiapoptotic (N-terminally truncated, ΔN) function. Unlike p53, the role of p73 and p63 in tumor is controversial. It has been recently hypothesized that altered ΔN:TA expression ratio, rather than single isoform overexpression, plays a role in the pathogenesis of many diseases, including lung cancer. Methods: Isoform-specific, real-time polymerase chain reaction and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLCs) have been performed aiming to explore the expression levels of each p63 and p73 N -terminal isoforms and their ΔN:TA expression ratio. Results: For both p63 and p73, a N -terminal isoform-specific modulation that alter ΔN:TA isoform balance was identified. In particular, ΔNp63 isoform was significantly up-modulated, whereas TAp63 was slightly down-modulated in NSCLC specimens. Likewise, Δ2p73 and Δ2/3p73 were up-modulated, whereas ΔNp73 and ΔN′p73 isoforms were down-modulated. Moreover, a higher TAp63 and ΔN′p73 transcripts expression, detected in the normal tissue surrounding the tumors, correlates with poor patient outcome, representing independent prognostic factors for overall survival (ΔN′p73: p = 0.049, hazard ratio = 3.091, 95% confidence interval = 1.005–9.524 and TAp63: p = 0.001, hazard ratio = 8.091, 95% confidence interval = 2.254–29.05). Conclusion: Our findings suggest that p63 and p73 altered ΔN:TA expression ratio occurs in NSCLC likely contributing to the molecular pathogenesis of this tumor.

To the Editor: Many anti‐cancer vaccination strategies have been tested in mice and humans in the attempt to eradicate leukemia cells 1. The vast majority of clinical trials are based on peptide vaccination which allows the induction of cellular response to specific tumor associated antigens 2. WT1(Wilms tumor‐1) gene is located on chromosome 11p13 and encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation. WT1 was originally described as a tumor suppressor gene although many evidences demonstrated that it plays an oncogenic function in the setting of leukemia. WT1 protein represents an optimal tumor antigen since it is highly expressed in acute leukemias, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms 3. By contrast, it is expressed at very low levels in normal hematopoietic progenitors. Expression of the WT1 protein is restricted to a limited set of tissues, including the gonads, uterus, kidney, and spleen.

Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL T315I mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation. The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors. We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

BACKGROUND The objective of this study was to evaluate the ability of the clinically available histone deacetylase (HDAC) inhibitor valproate to enhance the cytotoxicity of the Bcr-Abl inhibitor imatinib in imatinib-resistant cell lines. METHODS Interactions between imatinib, and valproate have been examined in imatinib-sensitive and -resistant chronic myeloid leukemia (CML)cell lines (K562, KCL-22, CML-T1) and in bone marrow mononuclear cells (MNCs) derived from imatinib-resistant CML patients. RESULTS In imatinib-sensitive cell lines, cotreatment with imatinib 0.5 μM and valproate 5 μM for 48 hours potently enhanced imatinib-induced growth arrest and apoptosis. In resistant cell lines and in primary MNCs derived from imatinib-resistant patients, valproate restored sensitivity to the cytotoxic effects of imatinib. Coexposure of cells to valproate and imatinib was associated with repression of several genes involved in Bcr-Abl transformation. In particular, the combination valproate–imatinib downregulated the expression of Bcr-Abl and the antiapoptotic protein Bcl-2, which is particularly overexpressed in imatinib-resistant clones. CONCLUSIONS Data from this study suggested that administration of the clinically available HDAC inhibitor valproate may be a powerful strategy to enhance cytotoxic effects of imatinib in those patient resistant to imatinib or in which complete cytogenetic remission has been not reached. Cancer 2006. © 2006 American Cancer Society.

Imatinib represents at present the most attractive therapy for BCR-ABL positive leukemias, even though a percentage of CML patients develop resistance to this compound. For these resistant patients a therapeutic approach based on a combination of drugs is more likely to be effective. In the last years, constitutive NF-kappaB/Rel activity has been demonstrated in several hematological malignancies. As a result, NFkB/Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-kappaB activation pathway offers a selective target to address tailored therapies. In the current study, we show that the IKK inhibitor PS1145 is able to inhibit the proliferation of CML cell lines and primary BM cells. Moreover, the addition of Imatinib increases the effects of PS1145 in resistant cell lines and BM cells from resistant patients, with a further increase of apoptosis and inhibition of proliferation and colony growth. Our data provide the rational for a new therapeutic approach, which combines Imatinib and the IKK inhibitor PS1145 in CML resistant patients.

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm 2 (SS5) or 15 dyne/cm 2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45±5.5 and 30±1.5 cells per field, respectively ( P 2 and also when SS of 5 and 15 dyne/cm 2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9±7 and 18±5 ng/10 6 cells for 16 hours, respectively ( P 6 cells for 16 hours, respectively ( P CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRα or PDGFRβ increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRα inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB ( P

Since the discovery of Ras, Ras-mediated transforming activity has been the major investigative area of interest. Soon thereafter it has emerged that Ras family members regulate different biological processes, other than cell growth, like development and fine-tune the balance between cell death and survival. The lower metazoan Dictyostelium discoideum is a powerful and genetically accessible model organism that has been used to elucidate the roles played by different Ras members in some biological processes, such as cell motility and development. In the following chapter we describe some very basic techniques aiming to identify novel Ras signaling components, throughout insertional mutagenesis screening, and to investigate their role(s) in development and chemotaxis processes.

Since the discovery of Ras, Ras-mediated transforming activity has been the major investigative area of interest. Soon thereafter it has emerged that Ras family members regulate different biological processes, other than cell growth, like development and fine-tune the balance between cell death and survival. The lower metazoan Dictyostelium discoideum is a powerful and genetically accessible model organism that has been used to elucidate the roles played by different Ras members in some biological processes, such as cell motility and development. In the following chapter we describe some very basic techniques aiming to identify novel Ras signaling components, throughout insertional mutagenesis screening, and to investigate their role(s) in development and chemotaxis processes.

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. TheracF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyosteliumlife cycle. Highest identity (94%) was found to a RacF2 isoform, toDictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell–cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell–cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.