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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

There is emerging evidence that, beyond their cholesterol-lowering properties, statins exhibit important antileukemic effects in vitro and in vivo, but the precise mechanisms by which they generate such responses remain to be determined. We have previously shown that statins promote differentiation of acute promyelocytic leukemia cells and enhance generation of all-trans retinoic acid (ATRA)–dependent antileukemic responses. We now provide evidence that statin-dependent leukemic cell differentiation requires engagement and activation of the c-Jun NH2-terminal kinase kinase pathway. In addition, in experiments, to define the molecular targets and mediators of statin-induced differentiation, we found a remarkable effect of statins on ATRA-dependent gene transcription, evidenced by the selective induction of over 400 genes by the combination of atorvastatin and ATRA. Altogether, our studies identify novel statin molecular targets linked to differentiation, establish that statins modulate ATRA-dependent transcription, and suggest that combined use of statins with retinoids may provide a novel approach to enhance antileukemic responses in acute promyelocytic leukemia and possibly other leukemias. [Mol Cancer Ther 2009;8(3):615–25]

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 from Regulation of leukemic cell differentiation and retinoid-induced gene expression by statins

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]

Genes involved in DNA repair and replication have been recently investigated as predictive markers of response to chemotherapy in non–small cell lung cancer (NSCLC). However, few data on the expression of these genes in tumor compared with corresponding normal lung are available. The aim of this study was to evaluate differential mRNA levels of 22 DNA repair genes of five different DNA repair pathways: direct, base excision, nucleotide excision (NER), double-strand break (DSBR), and postreplicative repair. In addition, six genes involved in DNA replication (REP) and three telomere maintenance genes were investigated. Total RNAs extracted from fresh-frozen tumors and corresponding normal tissues of 50 consecutive chemo-naïve resected NSCLC patients were analyzed. Transcript levels were quantified by real-time PCR. A significant overexpression was detected in 20 of 30 (67%) genes, mostly belonging to DSBR pathways, whereas others (XPA, XPC, and UBE2N; 10%) were significantly underexpressed. For 7 of 30 (23%) genes, mostly belonging to NER pathway, no significant difference between paired tumor and normal samples was observed. Transcript overexpression of DSBR and REP genes was significantly higher in poorly differentiated carcinomas and DSBR levels were higher in men compared with women. The transcriptional overexpression of four genes (XRCC5, TOP3B, TYMS, and UNG) showed significant correlation with a shorter patients' outcome at the univariate, whereas only stage of disease appeared as an independent factor affecting prognosis, as assessed by multivariate analysis. In conclusion, genes belonging to DNA repair/replication pathways are overexpressed in NSCLC and are associated with a more aggressive phenotype. [Cancer Res 2009;69(8):3390–6]

Supplementary Table 1 from Non–Small Cell Lung Cancer Exhibits Transcript Overexpression of Genes Associated with Homologous Recombination and DNA Replication Pathways

Studies have shown a link between the downregulation of connexin 43 (Cx43), the predominant isoform in cardiac gap junctions, and high susceptibility to cardiac arrhythmias and cardiomyocyte death. Non-myocytic cells (NMCs), the most abundant component of the heart, exert multiple cardiac functions and represent an important therapeutic target for diseased cardiac tissue. A few studies have investigated the effect of Apelin-13, an endogenous peptide with a key role in various cardiovascular functions, on Cx43 expression in cardiomyocytes. However, it remained unknown whether Apelin-13 influences Cx43 expression in NMCs. Here, we found that in NMCs, Cx43 protein expression increased after Apelin-13 treatment (100 nM for 48 h). Furthermore, dye transfer assays proved that Apelin-13-treated NMCs had a greater ability to communicate with surrounding cardiomyocytes, and this effect was abrogated by carbenoxolone, a gap junction inhibitor. Interestingly, we showed that Apelin-13 increased Cx43 through autophagy inhibition, as proved by the upregulation of p62 and LC3I, acting as 3-MA, a well-known autophagy inhibitor. In addition, Apelin-13-induced AKT and mTOR phosphorylation was abolished by LY294002 and rapamycin inhibitors resulting in Cx43 increased suppression. These results open the possibility of targeting gap junctions in NMCs with Apelin-13 as an exciting therapeutic approach with great potential.

Mutations in

Mutations in SF3B1 are found in 20% of myelodysplastic syndromes and 5–10% of myeloproliferative neoplasms, where they are considered important for diagnosis and therapy decisions. Sanger sequencing and NGS are the currently available methods to identify SF3B1 mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent SF3B1 mutation, p.Lys700Glu, we developed a novel fast and cheap assay based on PNA-PCR clamping. After setting the optimal PCR conditions, the limit of detection of PNA-PCR clamping was evaluated, and the method allowed up to 0.1% of mutated SF3B1 to be identified. Successively, PNA-PCR clamping and Sanger sequencing were used to blind test 90 DNA from patients affected by myelodysplastic syndromes and myeloproliferative neoplasms for the SF3B1 p.Lys700Glu mutation. PNA-PCR clamping and Sanger sequencing congruently identified 75 negative and 13 positive patients. Two patients identified as positive by PNA-PCR clamping were missed by Sanger analysis. The discordant samples were analyzed by NGS, which confirmed the PNA-PCR clamping result, indicating that these samples contained the SF3B1 p.Lys700Glu mutation. This approach could easily increase the characterization of myelodysplastic syndromes and myeloproliferative neoplasms in small-/medium-sized laboratories, and guide patients towards more appropriate therapy.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

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 Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and for postoperative adjuvant therapy. It is known that mitotane destroys the adrenal cortex impairing steroidogenesis, although its exact molecular mechanism is still unclear. However, confounding factors affecting in vitro experiments could reduce the relevance of the studies. In this review, we explore in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. On this basis, it may be necessary to re-evaluate the experiments taking into account their potential confounding factors such as cell strains, culture serum, lipoprotein concentration, and culture passages, which could hide important molecular results. As a consequence, the identification of novel pharmacological molecular pathways might be used in the future to implement personalized therapy, maximizing the benefit of mitotane treatment while minimizing its toxicity. Abstract Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and is increasingly used for postoperative adjuvant therapy. Mitotane action involves the deregulation of cytochromes P450 enzymes, depolarization of mitochondrial membranes, and accumulation of free cholesterol, leading to cell death. Although it is known that mitotane destroys the adrenal cortex and impairs steroidogenesis, its exact mechanism of action is still unclear. The most used cell models are H295-derived cell strains and SW13 cell lines. The diverging results obtained in presumably identical cell lines highlight the need for a stable in vitro model and/or a standard methodology to perform experiments on H295 strains. The presence of several enzymatic targets responsive to mitotane in mitochondria and mitochondria-associated membranes causes progressive alteration in mitochondrial structure when cells were exposed to mitotane. Confounding factors of culture affecting in vitro experiments could reduce the significance of any molecular mechanism identified in vitro. To ensure experimental reproducibility, particular care should be taken in the choice of culture conditions: aspects such as cell strains, culture serum, lipoproteins concentration, and culture passages should be carefully considered and explicated in the presentation of results. We aimed to review in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. If the concerns pointed out in this review will be overcome, the new insights into mitotane mechanism of action observed in-vitro could allow the identification of novel pharmacological molecular pathways to be used to implement personalized therapy.

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.

BCR-ABL1 fusion transcript is the minimal residual disease marker in chronic myeloid leukemia, 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT&ndash, PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients&rsquo, molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical BCR-ABL1 transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical BCR&ndash, ABL1 transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the BCR&ndash, ABL1/ABL1 level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical BCR&ndash, ABL1 patients, paving the foundation to include them in treatment-free remission protocols.

Objectives Non-small-cell-lung-cancer (NSCLC) in young adults (≤45 years-old) accounts for a very small proportion, as this disease usually occurs in people at older age. The youthful NSCLC may constitute an entity with different clinical-pathologic characteristics, having predominance of adenocarcinoma histology and affecting mostly non-smoker subjects. However, without specific guidelines, it is currently considered, both clinically and biologically, as the same disease of the older counterpart, although differences have been documented. Materials and methods Using formalin-fixed paraffin embedded diagnostic tissues (FFPE), targeted next-generation sequencing (NGS) technology allowed to provide insight the mutational pattern of 46 oncogenes and tumor-suppressor genes in 26 young patients (Y). Two additional populations, including a FFPE series of aged counterpart (A: 29 patients) and a group of healthy young controls (C: 21, blood provided), were also investigated to compare NGS profiles. Results Clinical features of enrolled young patients harmonized with literature data, being most of patients women (58%), never-smokers (38%) and with adenocarcinoma histology (96%). C group was adopted to filter all the non-synonymous genetic variations (NS-GVs) not-associated with malignant overt disease. This skimmed selection mostly highlighted three genes: TP53, EGFR and KRAS. TP53 NS-GVs were numerically more numerous in younger, many involving specific annotated hotspot (R248, R273, G245, R249 and R282); the majority of EGFR NS-GVs was detected in young patients, with higher allelic frequency and mostly represented by exon 19 deletions. On the contrary, KRAS NS-GVs were mainly detected in aged population, with a prevalent compact pattern involving p.G12 position and associated with adenocarcinoma histology. Conclusion This retrospective study confirmed the feasibility of NGS approach for genetic characterization of NSCLC young adult patients, supporting the involvement of TP53, EGFR, and KRAS alterations in the early onset of NSCLC. Some of these GVs, or their pattern, may potentially contribute to customized targeted therapies.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.

Background: Acute myeloid leukemia is a heterogeneous hematological disease, characterized by karyotypic and molecular alterations. Mutations in IDH2 have a role in diagnosis and as a minimal residue disease marker. Often the variant allele frequency during follow up is less than 20%, which represents the limit of detection of Sanger sequencing. Therefore, the development of sensitive methodologies to identify IDH2 mutations might help to monitor patients&rsquo, response to therapy. We compared three different methods to identify and monitor IDH2 mutations in patients&rsquo, specimens. Methods: Performances of PNA-PCR clamping, droplet digital PCR and Sanger for IDH2 status identification were evaluated and compared in 96 DNA patients&rsquo, specimens. Results: In contrast with Sanger sequencing, our results highlighted the concordance between PNA clamping and digital PCR. Furthermore, PNA-PCR clamping was able to detect more mutated DNA with respect to Sanger sequencing that showed several false negatives independently from the allelic frequency. Conclusions: We found that PNA-PCR clamping and digital PCR identified IDH2 mutations in DNA samples with comparable results in a percentage significantly higher compared to Sanger sequencing. PNA-PCR clamping can be used even in laboratories not equipped for sophisticated analyses, decreasing cost and time for IDH2 characterization.

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

// 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.

Introduction Mucin-rich lung adenocarcinomas (ADCs), namely mucinous and colloid ADCs, are classified as ADC variants according to the World Health Organization 2015 classification. A correlation between morphological patterns and mutational status of these rare entities is not well established. Methods We investigated the mutational profile of mucin-rich lung ADCs in correlation with histopathological and morphological features with the goal of identifying biological tumor characteristics of potential prognostic and therapeutic interest. A series of 54 surgically resected primary mucinous lung ADC samples were retrospectively analyzed for clinicopathological characteristics and by targeted next-generation sequencing. Results Fifty cases were invasive mucinous ADCs (32 pure and 18 mixed) and four were colloid-predominant ADCs. Invasive mucinous ADC cases with a pure mucinous pattern were associated with a lower risk of vascular invasion ( p = 0.01), absence of signet ring cells ( p = 0.03), negative nodal status ( p = 0.006), and early clinical stage ( p = 0.02). The most prevalent mutations involved the Kirsten rat sarcoma viral oncogene homolog gene ( KRAS ) and tumor protein p53 gene ( TP53 ). Most mutations clustered in the mitogen-activated protein/protein kinase B pathway and in the p53/DNA repair pathway. A few uncommon epidermal growth factor receptor gene ( EGFR ) mutations were found. A correlation between a higher number of mutations and favorable clinical outcome was seen ( p Conclusions Our data showed that mucinous ADCs have peculiar pathological and molecular features that might suggest the need for a differentially tailored therapeutic approach compared with that to conventional lung ADC.

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.

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.

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.

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.

Purpose: In non–small cell lung cancer, higher thymidylate synthase (TS) levels have been reported in squamous cell carcinoma (SCC) compared with adenocarcinoma (ADC). Data on TS expression in large-cell carcinoma (LCC) are scanty. Experimental Design: TS mRNA and protein levels were analyzed in 42 surgical cases of pulmonary LCC, including 8 large-cell neuroendocrine carcinomas, and were compared with controls represented by ADC (n = 41), SCC (n = 30), and small-cell lung carcinoma (SCLC; n = 33). TS levels were also correlated with the expression of Ki67 and E2F1. Moreover, the reliability of TS expression analysis was assessed in 22 matched cytologic and surgical specimens of non–small cell lung cancer. Results: TS mRNA levels of LCC were comparable with those of control SCC, but significantly higher than those of ADC (P < 0.001) and lower than SCLC (P < 0.001). A correlation between TS mRNA and protein levels was observed in control ADC and SCC, but not in LCC. Large-cell neuroendocrine carcinomas had the highest TS expression, whereas in non-neuroendocrine LCCs, TS protein levels were significantly higher (P = 0.02) in LCC immunoreactive for p63 and desmocollin3 (markers of squamous differentiation) than those expressing TTF-1 (a marker of ADC). Both E2F1 and Ki67 levels were not correlated with TS in LCCs. Finally, a linear correlation in TS protein levels was observed between matched cytologic and surgical specimens. Conclusion: The pulmonary LCC immunoprofile may resemble that of SCCs or ADCs. This immunoprofile is associated with differential TS expression levels, which may support a more appropriate therapeutic strategy decision. (Clin Cancer Res 2009;15(24):7547–52)

// Marco Lo Iacono 1, * , Consuelo Buttigliero 1, * , Valentina Monica 1, * , Enrico Bollito 1 , Diletta Garrou 1 , Susanna Cappia 1 , Ida Rapa 1 , Francesca Vignani 1 , Valentina Bertaglia 1 , Cristian Fiori 1 , Mauro Papotti 1 , Marco Volante 1 , Giorgio V. Scagliotti 1 , Francesco Porpiglia 1 , Marcello Tucci 1 1 University of Turin, Department of Oncology, Orbassano, Italy * All these Authors equally contributed to the experimental study Correspondence to: Consuelo Buttigliero, e-mail: consuelo.buttigliero@gmail.com Keywords: next generation sequencing, prostate cancer, precision medicine, genetic characterization, prognostic factors Received: November 17, 2015 Accepted: January 25, 2016 Published: February 12, 2016 ABSTRACT Purpose: Prostate cancer (PCa) has a highly heterogeneous outcome. Beyond Gleason Score, Prostate Serum Antigen and tumor stage, nowadays there are no biological prognostic factors to discriminate between indolent and aggressive tumors. The most common known genomic alterations are the TMPRSS-ETS translocation and mutations in the PI3K, MAPK pathways and in p53, RB and c-MYC genes. The aim of this retrospective study was to identify by next generation sequencing the most frequent genetic variations (GVs) in localized and locally advanced PCa underwent prostatectomy and to investigate their correlation with clinical-pathological variables and disease progression. Results: Identified non-synonymous GVs included TP53 p.P72R (78% of tumors), two CSFR1 SNPs, rs2066934 and rs2066933 (70%), KDR p.Q472H (67%), KIT p.M541L (28%), PIK3CA p.I391M (19%), MET p.V378I (10%) and FGFR3 p.F384L/p.F386L (8%). TP53 p.P72R, MET p.V378I and CSFR1 SNPs were significantly associated with the HI risk group, TP53 and MET variations with T≥T2c. FGFR3 p.F384L/p.F386L was correlated with T≤T2b. MET p.V378I mutation, detected in 20% of HI risk patients, was associated with early biochemical recurrence. Experimental design: Nucleic acids were obtained from tissue samples of 30 high (HI) and 30 low-intermediate (LM) risk patients, according to D’Amico criteria. Genomic DNA was explored with the Ion_AmpliSeq_Cancer_Hotspot_Panel_v.2 including 50 cancer-associated genes. GVs with allelic frequency (AF) ≥10%, affecting protein function or previously associated with cancer, were correlated with clinical-pathological variables. Conclusion: Our results confirm a complex mutational profile in PCa, supporting the involvement of TP53, MET, FGFR3, CSF1R GVs in tumor progression and aggressiveness.

Membrane-derived microvesicles (MVs) are released from the cell surface and are implicated in cell-to-cell communication. We evaluated whether MVs derived from endothelial progenitor cells (EPCs) are able to trigger angiogenesis. We found that EPC-derived MVs were incorporated in endothelial cells by interaction with α4 and β1 integrins expressed on the MV surface. In vitro, MVs promoted endothelial cell survival, proliferation, and organization in capillary-like structures. In vivo, in severe combined immunodeficient (SCID) mice, MV-stimulated human endothelial cells organized in patent vessels. When incubated with RNase, despite their internalization into endothelial cells, MVs failed to induce in vitro and in vivo angiogenic effects. mRNA transfer was shown by transduction of GFP protein in endothelial cells by MVs containing GFP-mRNA and the biologic relevance by the angiogenic effect of MV-mRNA extract delivered by lipofectamine. Microarray ana-lysis and quantitative reverse transcription–polymerase chain reaction (RT-PCR) of MV-mRNA extract indicated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated with the PI3K/AKT signaling pathway. Protein expression and functional studies showed that PI3K and eNOS play a critical role in the angiogenic effect of MVs. These results suggest that EPCs may activate angiogenesis in endothelial cells by releasing MVs able to trigger an angiogenic program.

Purpose Non–small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. Patients and Methods A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. Results Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). Conclusion The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

The combination of cytotoxic chemotherapy with signaling pathway inhibitors represents a potential strategy to improve the treatment of nonsmall cell lung cancer (NSCLC). Thymidylate synthase (TS) is an enzyme essential for DNA synthesis, and its overexpression has been associated with the reduced sensitivity to antifolate agents. Src is a tyrosine kinase that modulates the cytotoxicity of cancer cells after drug treatment, and in vitro data indicate that its inhibition could revert the resistance to TS-inhibiting drugs. Our study investigated the significance of TS and Src expression in NSCLC tissues, and the effects of their pharmacological inhibition in cell lines. In tumor and normal tissues from 94 resected NSCLC patients, TS and Src transcript levels were found positively correlated (RS = 0.66), associated with patients smoking history and overall survival. At multivariate analysis, TS gene expression was an independent prognostic factor (relative risk (RR) = 1.78, from 1.16 to 2.72; p < 0.01). Immunohistochemical detection in tumor specimens confirmed that Src kinase activation, evaluated by phospho-specific antibody, was associated to a higher TS expression. In cell lines, dasatinib, a Src-inhibiting agent, synergistically enhanced pemetrexed-cytotoxicity of A549 cells, as evaluated by MTT and apoptosis assays. The biological explanation for this interaction was based on the upregulation of TS messenger RNA and protein levels induced by pemetrexed, which was significantly prevented by dasatinib cotreatment. The data of our study suggest that TS and Src may belong to a common pathway that bears prognostic significance in NSCLC, and that Src represents a potential target to improve the efficacy of TS-inhibiting agents.

Background Aurora kinase A (AURKA) is a member of serine/threonine kinase family. Several kinases belonging to this family are activated in the G2/M phase of the cell cycle being involved in mitotic chromosomal segregation. AURKA overexpression is significantly associated with neoplastic transformation in several tumors and deregulated Aurora Kinases expression leads to chromosome instability, thus contributing to cancer progression. The purpose of the present study was to investigate the expression of AURKA in non small cell lung cancer (NSCLC) specimens and to correlate its mRNA or protein expression with patients' clinico-pathological features. Materials and methods Quantitative real-time PCR and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLC) have been performed aiming to explore the expression levels of AURKA gene. Results AURKA expression was significantly up-modulated in tumor samples compared to matched lung tissue (p < 0.01, mean log2(FC) = 1.5). Moreover, AURKA was principally up-modulated in moderately and poorly differentiated lung cancers (p < 0.01), as well as in squamous and adenocarcinomas compared to the non-invasive bronchioloalveolar histotype (p = 0.029). No correlation with survival was observed. Conclusion These results indicate that in NSCLC AURKA over-expression is restricted to specific subtypes and poorly differentiated tumors.

Genes involved in DNA repair and replication have been recently investigated as predictive markers of response to chemotherapy in non–small cell lung cancer (NSCLC). However, few data on the expression of these genes in tumor compared with corresponding normal lung are available. The aim of this study was to evaluate differential mRNA levels of 22 DNA repair genes of five different DNA repair pathways: direct, base excision, nucleotide excision (NER), double-strand break (DSBR), and postreplicative repair. In addition, six genes involved in DNA replication (REP) and three telomere maintenance genes were investigated. Total RNAs extracted from fresh-frozen tumors and corresponding normal tissues of 50 consecutive chemo-naïve resected NSCLC patients were analyzed. Transcript levels were quantified by real-time PCR. A significant overexpression was detected in 20 of 30 (67%) genes, mostly belonging to DSBR pathways, whereas others (XPA, XPC, and UBE2N; 10%) were significantly underexpressed. For 7 of 30 (23%) genes, mostly belonging to NER pathway, no significant difference between paired tumor and normal samples was observed. Transcript overexpression of DSBR and REP genes was significantly higher in poorly differentiated carcinomas and DSBR levels were higher in men compared with women. The transcriptional overexpression of four genes (XRCC5, TOP3B, TYMS, and UNG) showed significant correlation with a shorter patients' outcome at the univariate, whereas only stage of disease appeared as an independent factor affecting prognosis, as assessed by multivariate analysis. In conclusion, genes belonging to DNA repair/replication pathways are overexpressed in NSCLC and are associated with a more aggressive phenotype. [Cancer Res 2009;69(8):3390–6]

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]

There is emerging evidence that, beyond their cholesterol-lowering properties, statins exhibit important antileukemic effects in vitro and in vivo, but the precise mechanisms by which they generate such responses remain to be determined. We have previously shown that statins promote differentiation of acute promyelocytic leukemia cells and enhance generation of all-trans retinoic acid (ATRA)–dependent antileukemic responses. We now provide evidence that statin-dependent leukemic cell differentiation requires engagement and activation of the c-Jun NH2-terminal kinase kinase pathway. In addition, in experiments, to define the molecular targets and mediators of statin-induced differentiation, we found a remarkable effect of statins on ATRA-dependent gene transcription, evidenced by the selective induction of over 400 genes by the combination of atorvastatin and ATRA. Altogether, our studies identify novel statin molecular targets linked to differentiation, establish that statins modulate ATRA-dependent transcription, and suggest that combined use of statins with retinoids may provide a novel approach to enhance antileukemic responses in acute promyelocytic leukemia and possibly other leukemias. [Mol Cancer Ther 2009;8(3):615–25]

Background In real-time RT quantitative PCR (qPCR) the accuracy of normalized data is highly dependent on the reliability of the reference genes (RGs). Failure to use an appropriate control gene for normalization of qPCR data may result in biased gene expression profiles, as well as low precision, so that only gross changes in expression level are declared statistically significant or patterns of expression are erroneously characterized. Therefore, it is essential to determine whether potential RGs are appropriate for specific experimental purposes. Aim of this study was to identify and validate RGs for use in the differentiation of normal and tumor lung expression profiles. Methods A meta-analysis of lung cancer transcription profiles generated with the GeneChip technology was used to identify five putative RGs. Their consistency and that of seven commonly used RGs was tested by using Taqman probes on 18 paired normal-tumor lung snap-frozen specimens obtained from non-small-cell lung cancer (NSCLC) patients during primary curative resection. Results The 12 RGs displayed showed a wide range of Ct values: except for rRNA18S (mean 9.8), the mean values of all the commercial RGs and ESD ranged from 19 to 26, whereas those of the microarray-selected RGs (BTF-3, YAP1, HIST1H2BC, RPL30) exceeded 26. RG expression stability within sample populations and under the experimental conditions (tumour versus normal lung specimens) was evaluated by: (1) descriptive statistic; (2) equivalence test; (3) GeNorm applet. All these approaches indicated that the most stable RGs were POLR2A, rRNA18S, YAP1 and ESD. Conclusion These data suggest that POLR2A, rRNA18S, YAP1 and ESD are the most suitable RGs for gene expression profile studies in NSCLC. Furthermore, they highlight the limitations of commercial RGs and indicate that meta-data analysis of genome-wide transcription profiling studies may identify new RGs.

p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome, ADULT (acro, dermato, ungual, lacrimal, tooth) syndrome and AEC (ankyloblepharon, ectodermal dysplasia, clefting) syndrome (also called Hay-Wells syndrome). The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. In this paper we report a study on the TAp63alpha isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63alpha wt, DeltaNp63alpha or the TAp63alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63alpha and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.

Abstract 2171 Poster Board II-148 Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks. Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood. The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively. CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L). The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis. Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors. Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA. By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam-68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex pre-mRNA splicing scenario. Disclosures: No relevant conflicts of interest to declare.

Background: Mutations occurring within the kinase domain of the tyrosine kinase c-Abl, and its oncogenic counterpart Bcr-Abl, strongly affects the outcome of clinical treatment with tyrosine kinase inhibitors (TKIs). Although a second and third generation of TKIs has been developed and these molecules are currently available some mutations (i.e. T315I) confer insensitivity to the drugs. Therefore, a method allowing quick detection of these mutations still represents a challenge for most of clinicians because it would allow them to rapidly switch to alternative therapies instead of TKIs. Preliminary data of a novel method allowing easy and rapid detection of the mutation affecting the codon 315 (T315I), which is nowadays the most severe and resistant mutation to TKIs, is presented. Rationale & Methods: The innovative strategy we propose is based on a variant of a single tube PNA-PCR clamping. Peptide Nucleic Acid (PNA) are synthetic molecules sharing physical-chemical features with DNA. Indeed, they possesses a structural backbone formed by N-(2-amino-ethyl)-glycine, instead of deoxy-ribose molecules linked together by phosphodiester bond, covalently conjugated with the four bases (A, T, G, C). This peculiar structure confers to the molecule exceptionally high stability when matches a single complementary DNA strand. Interestingly, the binding of PNA to nucleic acids is not affected by ionic strength and, compared to identical homo-duplex DNA-DNA, displays higher melting temperature (Tm). The latter feature allows detection of point mutations because a single mismatch within the sequences would lower the Tm of approximately 20°C. Taking advantage of these unique PNA properties, together with the fact that PNA can not prime DNA polymerase job, we utilized sequence specific PNA to block PCR amplification. The approach makes use of a specific clamping mechanism, the “primer exclusion”, in which PNA and oligonucleotide primer compete for the same target sequence. As consequence when PNA binds to template DNA the DNA polymerase can not synthesize new DNA strand. The experimental design we propose schedules the use of a PNA harboring a sequence which perfectly matches within the wild-type (wt) c-Abl tyrosine kinase domain. Therefore, when a single base pair mismatch occurs, such as it happens in presence of mutation conferring TKIs resistence, the hetero-duplex PNA-DNA stability is strongly impaired. The expected PCR clamping readout would be as follow: in case of wt template PCR reaction will not work because DNA polymerase activity is impaired by the formation of PNA-DNA hetero-duplex; on the other hand when a mutation occurs the stability between PNA and DNA complementary sequence is compromised allowing oligonucleotide primer to displace PNA. So far we focused our efforts on the mutation affecting codon 315 (T315I). Results & Conclusions: cDNA synthesized from total RNA isolated from patients harboring either wt or mutated Bcr-Abl alleles have been amplified by nested PCR. During the first step a specific pair of primers designed to discriminate between the kinase domain of c-Abl and Bcr-Abl have been utilized. The second step involves the use of sequence specific 19-mer PNA matching within the region around the codon 315 (T315I), so far the most severe mutation described, in a “PNA primer exclusion” PCR clamping reaction. Initially, we aimed to study patients which have previously been analyzed by direct sequencing in which either the wt or the mutated allele were to be predominant. Encouraging results show amplification in patients harboring the point mutation at position 315 which determine the substitution of a threonine for isoleucine (T315I). These results were further corroborated by carrying-out a blind analysis, in which samples were subsequently sequenced. These data show that, in future, the technique herein reported might be extremely useful in detecting the presence of all known mutations within the Bcr-Abl kinase domain with simple, rapid, reliable and very cheap methodology.

Introduction Tyrosine kinases (TKs) play different roles either in physiological or in pathological cellular processes ranging from the regulation of intracellular signaling pathways leading for example to proliferation, differentiation and/or DNA syntheisis. The whole human genome encodes for approximately 95 TKs classified in two major families: transmembrane and non-transmembrane TKs. Transmembrane TKs are further splitted in two groups: Receptor Tyrosine Kinases (RTKs) and Non-Receptor Transmembrane Tyrosine Kinases. Despite in the last decade the role of RTKs has been deeply investigated very little is known about the second class of TKs, Non-Receptor Transmembrane Tyrosine Kinases. Recently, we began to investigate the roles played by these peculiar molecules in hematological tissues and preliminary data are herein presented and discussed. Material & Methods and Results STYK1 is a Non-Receptor Transmembrane TK characterized by a very short extracellular domain, of approximately 25 aminoacid residues followed by a single transmembrane alpha-helix sequence and a conserved cytoplasmic region harboring the catalytic domain, splitted in two halves separated by a short kinase insert sequence, peculiar of class III RTKs, VEGFR and FGFR family members. Indeed, sequence analysis alignment reveals the higher homology with PDGFR and FGFR members. Surprisingly, data mining searches, performed with the Ensembl tools (http://www.ensembl.org) and using blast algorithm with human STYK1 protein as query, revealed that this protein possesses orthologues either in vertebrate model organisms, both mammalian and non-mammalian or in invertebrate metazoan, such as Ciona. Furthermore, a bioinformatic analysis carried-out at CBS (http://www.cbs.dtu.dk/services/NetPhos/) revealed 4 tyrosine residues putatively phosphorylated. Interestingly one of the tyrosine residue displays high homology towards a consensus sequence for the p85 regulatory subunit of the PI3K. Since our interest aims to understand the roles played by STYK1 in hemopoietic cells, we first investigated its expression in different blood cells populations. Therefore the different blood cellular components were separated by immunoaffinity sorting and total mRNA was extracted. Afterwards c-DNA was synthesized and STYK1 mRNA expression studied by RT-PCR. The results reveal that STYK1 mRNA is expressed predominantly in mature lymphocytes. To study more in details its expression within lymphoid population we attempted to separate B-from T-lymphocytes and STYK1 mRNA expression evaluated in the two populations. Furthermore, we also investigated STYK1 expression in leukemic hematological disorders. Nevertheless STYK1 mRNA expression is typical of mature hemopoietic cells populations, such as lymphocytes, its expression resulted deregulated in patients affected by different kind of leukemia (i.e. CML, ALL, AML). Currently a constitutively active form of STYK1 has been generated and its signaling is under investigation. Conclusion The non-receptor transmembrane tyrosine kinase STYK1 represents a novel lymphocyte molecular marker whose expression results deregulated in patients affected by leukemia. Interestingly, due to its homology with class III RTKs like PDGFR, which are easily targeted by small molecules such as Imatinib mesylate, it may represents a novel druggable molecular target for leukemic cells.

22017 Background: Genes involved in DNA repair and/or replication have been recently investigated as predictive markers of response to chemotherapy in NSCLC. However, few data on the expression of these genes in NSCLC tumor samples versus corresponding normal lung are currently available. Aim of this study was to evaluate differential mRNA levels of 22 DNA repair genes belonging to 5 different DNA repair pathways: direct (DR), base excision (BER), nucleotide excision (NER), double strand break (DSBR) and post-replicative (PRR) repair. In addition, 6 genes involved in DNA replication (REP) and 2 telomere maintenance (TM) genes were also investigated. Methods: Total RNA was extracted from fresh snap-frozen tumors and corresponding normal tissues from 50 consecutive chemo-naive resected NSCLC patients. Transcript levels were quantified by Real-Time PCR (TaqMan), fold changes were calculated with the 2-ΔΔCt method and statistical significance assessed by Wilcoxon's test. POLR2A and 18SrRNA were used as refere...

IntroductionMalignant pleural mesothelioma (MPM) is a rare malignant disease, and the understanding of molecular pathogenesis has lagged behind other malignancies.MethodsA series of 123 formalin-fixed, paraffin-embedded tissue samples with clinical annotations were retrospectively tested with a commercial library kit (Ion AmpliSeq Cancer Hotspot Panel v.2, Life Technologies, Grand Island, NY) to investigate 50 genes plus other two, BRCA1-associated protein-1 (BAP-1) and neurofibromatosis-2 (NF2), frequently altered in MPM. DNA was obtained from tissues after manual microdissection and enriched for at least 50% cancer cells. Variations affecting protein stability or previously correlated to cancer, more frequently identified (≥25 patients with at least 10% of allelic frequency), were subsequently evaluated by Sanger sequencing. Immunohistochemistry staining for BAP1 and NF2 proteins was also performed.ResultsThe commonest genetic variations were clustered in two main pathways: the p53/DNA repair (TP53, SMACB1, and BAP1) and phosphatidylinositol 3-kinase–AKT pathways (PDGFRA, KIT, KDR, HRAS, PIK3CA, STK11, and NF2). PIK3CA:c.1173A>G mutation, STK11:rs2075606 (T>C), or TP53:rs1042522 (Pro/Pro) was significantly associated with time to progressive disease (TTPD; all p values < 0.01). Furthermore, the accumulation of genetic alterations correlated with shorter TTPD and reduced overall survival (TTPD p value = 0.02, overall survival p value = 0.04). BAP1 genetic variations identified were mainly located in exons 13 and 17, and BAP1 nonsynonymous variations were significantly correlated with BAP1 protein nuclear localization.ConclusionNext-generation sequencing was applied to a relatively large retrospective series of MPM using formalin-fixed, paraffin-embedded archival material. Our results indicate a complex mutational landscape with a higher number of genetic variations in the p53/DNA repair and phosphatidylinositol 3-kinase pathways, some of them with prognostic value.