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Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30-40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.

Patients on extracorporeal support for severe acute respiratory distress syndrome may require a prolonged period of deep sedation. In these patients, volatile sedation may represent a valid alternative to IV drugs. The aim of our study was to describe the feasibility of volatile sedation in a large cohort of acute respiratory distress syndrome patients undergoing venovenous extracorporeal membrane oxygenation and ultraprotective ventilation., Design: Retrospective monocentric study., Setting: Adult ICU, ASST Monza, Italy., Patients: Adult patients who underwent volatile sedation with isoflurane during venovenous extracorporeal membrane oxygenation between 2009 and 2019., Interventions: Isoflurane was delivered via the AnaConDa system. The sedation level, hemodynamics, and laboratory tests were compared between the volatile sedation phase and the IV sedation phases before and after the isoflurane sedation period., Measurements and Main Results: About 74 patients (50 yr [43-56 yr]) were included. Median duration of venovenous extracorporeal membrane oxygenation support was 22 days (14-51 d). Volatile sedation started on day 3 (2-6) of extracorporeal membrane oxygenation support, and its median duration was 7 days (4-13 d), ranging from 1 to 38 days. A total of 970 venovenous extracorporeal membrane oxygenation days were analyzed. During the volatile phase, the sedation level was slightly deeper (bispectral index 39 ± 6) compared with the IV phase before and after isoflurane (42 ± 8 and 43 ± 9, respectively, p < 0.001). Requirements of fentanyl and remifentanyl were reduced during the volatile phase. Minor differences in hemodynamics were observed during volatile sedation: mean arterial pressure was lower (75 ± 13 vs 79 ± 14 and 80 ± 15; p < 0.001), whereas cardiac output was higher (8.5 ± 1.9 vs 7.9 ± 1.8 and 8.0 ± 1.8; p = 0.003). Aspartate aminotransferase levels were lower during the volatile sedation phases ( p < 0.001), whereas alanine aminotransferase, triglycerides, and creatine phosphokinase were more altered during the IV sedation phase before isoflurane ( p < 0.001)., Conclusions: Volatile sedation represents an alternative to IV agents to achieve long-term deep sedation in critically ill patients on extracorporeal membrane oxygenation undergoing ultraprotective ventilation., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Pharmacological cancer therapy is often based on the concurrent inhibition of different survival pathways to improve treatment outcomes and to reduce the risk of relapses. While this strategy is traditionally pursued only through the co-administration of several drugs, the recent development of multi-targeting drugs (i.e., compounds intrinsically able to simultaneously target several macromolecules involved in cancer onset) has had a dramatic impact on cancer treatment. This review focuses on the most recent developments in dual-kinase inhibitors used in acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and lymphoid tumors, giving details on preclinical studies as well as ongoing clinical trials. A brief overview of dual-targeting inhibitors (kinase/histone deacetylase (HDAC) and kinase/tubulin polymerization inhibitors) applied to leukemia is also given. Finally, the very recently developed Proteolysis Targeting Chimeras (PROTAC)-based kinase inhibitors are presented.

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1 -negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1 , ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1 , SETBP1 , and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival., Competing Interests: The authors declare no competing interests., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN 1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib., (©2018 American Association for Cancer Research.)

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel-Giedion syndrome caused by SETBP1 mutations.

The rearranged during transfection ( RET ) proto-oncogene was recognized as the multiple endocrine neoplasia type 2 (MEN2) causing gene in 1993. Since then, much effort has been put into a clear understanding of its oncogenic signaling, its biochemical function and ways to block its aberrant activation in MEN2 and related cancers. Several small molecules have been designed, developed or redirected as RET inhibitors for the treatment of MEN2 and sporadic MTC. However, current drugs are mostly active against several other kinases, as they were not originally developed for RET. This limits efficacy and poses safety issues. Therefore, there is still much to do to improve targeted MEN2 treatments. New, more potent and selective molecules, or combinatorial strategies may lead to more effective therapies in the near future. Here, we review the rationale for RET targeting in MEN2, the use of currently available drugs and novel preclinical and clinical RET inhibitor candidates., (© 2018 Society for Endocrinology.)

Daily nursing in critical care patients may alter vital parameters, especially in the most critically ill patients. The aim of our study was to evaluate feasibility and safety of daily nursing on patients undergoing venous-venous extracorporeal membrane oxygenation (vv-ECMO) for severe respiratory failure. Daily nursing was performed following defined phases (sponge bath, elevation with scooping stretcher, change position of endotracheal tube, dressing replacement). We recorded physiological and ECMO parameters before and during daily nursing in 5 patients for several days (total: 25 daily nursing) and adverse events: desaturation, hypertension, reduction of mixed venous oxygen saturation, arterial oxygen saturation or ECMO blood flow and elevation in minute ventilation. Sedative drug dosage and additional bolus were recorded. Daily nursing was performed in 92 % of cases (23/25), with a minimum of two adverse events per daily nursing. Hypertension and tachycardia were mostly recorded at the beginning, while desaturation, reduction in mixed venous oxygen saturation and blood flow were recorded during elevation with scooping stretcher. Increase in minute ventilation was frequent in spontaneous breathing patients. Additional bolus of sedation was required before and/or during nursing. Daily nursing significantly alters physiologic parameters; thus, it should be performed only when physicians are readily available to treat adverse events.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/mTOR, indispensable for survival of ALK-driven tumors.Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while long-term exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR-ABL1 by Q-RT-PCR does not equate to eradication of the disease. Digital-PCR (dPCR), able to detect 1 BCR-ABL1 positive cell out of 10(7) , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q-RT-PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q-RT-PCR for 36 months. Patients losing molecular remission (two consecutive positive Q-RT-PCR with at least 1 BCR-ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow-up of 21.6 months. Fifty-two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not-relapsed patients, 40 (37.0% of total) regained Q-RT-PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013-1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients <45 years relapsed versus 42% in the class ≥45 to <65 years and 33% of patients ≥65 years [P(χ(2) ) < 0.0001]. Relapse rates ranged between 100% (<45 years, dPCR+) and 36% (>45 years, dPCR-). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse., (© 2015 Wiley Periodicals, Inc.)

Tumor suppressor function can be modulated by subtle variation of expression levels, proper cellular compartmentalization and post-translational modifications, such as phosphorylation, acetylation and sumoylation. The non-genomic loss of function of tumor suppressors offers a challenging therapeutic opportunity. The reactivation of a tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells. The identification of mechanisms that affect tumor suppressor functions is therefore essential. In this work, we show that BCR-ABL promotes the accumulation of the NFKBIA gene product, IκBα, in the cytosol through physical interaction and stabilization of the protein. Furthermore, BCR-ABL/IκBα complex acts as a scaffold protein favoring p53 nuclear exclusion. We therefore identify a novel BCR-ABL/IκBα/p53 network, whereby BCR-ABL functionally inactivates a key tumor suppressor.

Background: Bosutinib is a recently approved ABL inhibitor. In spite of the well-documented effectiveness of BCR-ABL inhibitors in treating chronic myeloid leukemia, development of resistance is a continuous clinical challenge. Transporters that facilitate drug uptake and efflux have been proposed as one potential source of resistance to tyrosine kinase inhibitor treatment. Our aim was to determine which carriers are responsible for bosutinib transport., Methods: K562S cells overexpressing the drug transporters ABCB1, ABCG2, and SLC22A1 were generated, characterized and used in proliferation assay and intracellular uptake and retention assay (IUR). In vivo experiments were performed in nude mice injected with K562S, K562DOX cells (overexpressing ABCB1), and K562DOX silenced for ABCB1 (K562DOX/sh P-GP)., Results: The IUR assay using C-14 bosutinib showed that only ABCB1 was responsible for active bosutinib transport. K562DOX cells showed the lowest intracellular level of bosutinib, while K562DOX cells treated with the ABCB1 inhibitor verapamil showed intracellular bosutinib levels comparable with parental K562S. Proliferation assays demonstrated that K562DOX are resistant to bosutinib treatment while verapamil is able to restore the sensitivity to the drug. Nude mice injected with K562DOX and treated with bosutinib showed very limited response and quickly relapsed after stopping treatment while K562S as well as K562DOX/sh P-GP remained tumor-free., Conclusions: Our data suggest that the analysis of ABCB1 expression levels might help determine treatment options for patients exhibiting resistance to bosutinib.

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders., (© 2015 by The American Society of Hematology.)

Background: Extracorporeal (EC) carbon dioxide (CO(2)) removal (ECCO(2)R) may be a powerful alternative to ventilation, possibly avoiding the need for mechanical ventilation and endotracheal intubation. We previously reported how an infusion of lactic acid before a membrane lung (ML) effectively enhances ECCO(2)R. We evaluated an innovative ECCO(2)R technique based on ventilation of acidified dialysate., Methods: Four swine were sedated, mechanically ventilated, and connected to a venovenous dialysis circuit (blood flow, 250 ml/min). The dialysate was recirculated in a closed loop circuit including a ML (gas flow, 10 liters/min) and then returned to the dialyzer. In each animal, 4 different dialysis flows (DF) of 200, 400, 600, and 800 ml/min were evaluated with and without lactic acid infusion (2.5 mEq/min); the sequence was completed 3 times. At the end of each step, we measured the volume of CO(2)R by the ML (V(co2)ML) and collected blood and dialysate samples for gas analyses., Results: Acid infusion substantially increased V(co2)ML, from 33 ± 6 ml/min to 86 ± 7 ml/min. Different DFs had little effect on V(co2)ML, which was only slightly reduced at DF 200 ml/min. The partial pressure of CO(2) of blood passing through the dialysis filter changed from 60.9 ± 3.6 to 37.1 ± 4.8 mm Hg without acidification and to 32.5 ± 5.3 mm Hg with acidification, corresponding to a pH increase of 0.18 ± 0.03 and 0.03 ± 0.04 units, respectively., Conclusions: Ventilation of acidified dialysate efficiently increased ECCO(2)R of an amount corresponding to 35% to 45% of the total CO(2) production of an adult man from a blood flow as low as 250 ml/min., (Copyright © 2014 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Extracorporeal carbon dioxide removal has been proposed to achieve protective ventilation in patients at risk for ventilator-induced lung injury. In an acute study, the authors previously described an extracorporeal carbon dioxide removal technique enhanced by regional extracorporeal blood acidification. The current study evaluates efficacy and feasibility of such technology applied for 48 h., Methods: Ten pigs were connected to a low-flow veno-venous extracorporeal circuit (blood flow rate, 0.25 l/min) including a membrane lung. Blood acidification was achieved in eight pigs by continuous infusion of 2.5 mEq/min of lactic acid at the membrane lung inlet. The acid infusion was interrupted for 1 h at the 24 and 48 h. Two control pigs did not receive acidification. At baseline and every 8 h thereafter, the authors measured blood lactate, gases, chemistry, and the amount of carbon dioxide removed by the membrane lung (VCO2ML). The authors also measured erythrocyte metabolites and selected cytokines. Histological and metalloproteinases analyses were performed on selected organs., Results: Blood acidification consistently increased VCO2ML by 62 to 78%, from 79 ± 13 to 128 ± 22 ml/min at baseline, from 60 ± 8 to 101 ± 16 ml/min at 24 h, and from 54 ± 6 to 96 ± 16 ml/min at 48 h. During regional acidification, arterial pH decreased slightly (average reduction, 0.04), whereas arterial lactate remained lower than 4 mEq/l. No sign of organ and erythrocyte damage was recorded., Conclusion: Infusion of lactic acid at the membrane lung inlet consistently increased VCO2ML providing a safe removal of carbon dioxide from only 250 ml/min extracorporeal blood flow in amounts equivalent to 50% production of an adult man.

Anaplastic lymphoma kinase (ALK)-positive lymphomas respond to chemotherapy, but relapses, which bear a poor prognosis, occur. Crizotinib inhibits ALK in vitro and in vivo and was administered as monotherapy to 11 ALK+ lymphoma patients who were resistant/refractory to cytotoxic therapy. The overall response rate was 10 of 11 (90.9%; 95% confidence interval [CI] = 58.7% to 99.8%). Disease status at the latest follow-up is as follows: four patients are in complete response (CR) (months >21, >30, >35, >40) under continuous crizotinib administration; 4 patients had progression of disease (months 1, 2, 2, 2); 1 patient obtained CR on crizotinib, received an allogeneic bone marrow transplant, and is in CR; 2 patients (treated before and/or after allogeneic bone marrow transplant) obtained and are still in CR but they have stopped crizotinib. Overall and progression-free survival rates at 2 years are 72.7% (95% CI = 39.1% to 94.0%) and 63.7% (95% CI = 30.8% to 89.1%), respectively. ALK mutations conferring resistance to crizotinib in vitro could be identified in relapsed patients. Crizotinib exerted a potent antitumor activity with durable responses in advanced, heavily pretreated ALK+ lymphoma patients, with a benign safety profile.

Introduction: Blood acidification by lactic acid infusion converts bicarbonate to CO2. This effect can be exploited to increase the transmembrane PCO2 gradient of an extracorporeal membrane lung, resulting in a significant increase of extracorporeal CO2 removal. Lactic acid, however, is an energetic substrate and its metabolism might increase total body CO2 production (VCO2), limiting the potential beneficial effects of this technique. The aim of our study was to compare VCO2 during isocaloric infusion of lactic acid or glucose., Methods: Six pigs (45 ± 5 kg) were sedated and mechanically ventilated. Estimated caloric needs were 2,300-2,400 Kcal/die (95 to 100 Kcal/h). A sequence of two steps lasting four hours each was performed: 1) Glucose, 97 kcal/h were administered as 50% glucose solution, and 2) Lactic Acid, approximately 48.5 kcal/h were administered as lactic acid and approximately 48.5 kcal/h as 50% glucose solution. This sequence was repeated three times with two-hour intervals. Every hour VCO2, arterial blood gases and lactate were measured. Blood glucose level was kept constant by titrating an insulin infusion, ventilation was adjusted to maintain arterial PCO2 at 50 mmHg, a normal value for our animal model., Results: During Lactic Acid steps VCO2 increased less than 5% compared to the Glucose steps (282 vs. 269 ml/min, P < 0.05); blood glucose did not differ between the two groups (respectively 101 ± 12 vs. 103 ± 8 mg/dl). Arterial lactate was always lower than 3 mmol/L. Arterial pH was lower during Lactic Acid steps (7.422 vs. 7.445, P < 0.05)., Conclusions: Replacing 50% of the caloric input with lactic acid increased total CO2 production by less than 5% compared to an equal caloric load provided entirely by a 50% glucose solution.

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM&#95;000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM&#95;002072.2:c.735+1C>T), ABCC 3 (NM&#95;003786.3:c.1783-1G>A), KLHDC 1 (NM&#95;172193.1:c.568-2A>G), HOOK 1 (NM&#95;015888.4:c.1662-1G>A), SMAD 9 (NM&#95;001127217.2:c.1004-1C>T), and DNAH 9 (NM&#95;001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC 3, HOOK 1. In ABCC 3 and HOOK 1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK 1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4-6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis.

Copy number alterations (CNA) are common events occurring in leukaemias and solid tumors. Comparative Genome Hybridization (CGH) is actually the gold standard technique to analyze CNAs; however, CGH analysis requires dedicated instruments and is able to perform only low resolution Loss of Heterozygosity (LOH) analyses. Here we present CEQer (Comparative Exome Quantification analyzer), a new graphical, event-driven tool for CNA/allelic-imbalance (AI) coupled analysis of exome sequencing data. By using case-control matched exome data, CEQer performs a comparative digital exonic quantification to generate CNA data and couples this information with exome-wide LOH and allelic imbalance detection. This data is used to build mixed statistical/heuristic models allowing the identification of CNA/AI events. To test our tool, we initially used in silico generated data, then we performed whole-exome sequencing from 20 leukemic specimens and corresponding matched controls and we analyzed the results using CEQer. Taken globally, these analyses showed that the combined use of comparative digital exon quantification and LOH/AI allows generating very accurate CNA data. Therefore, we propose CEQer as an efficient, robust and user-friendly graphical tool for the identification of CNA/AI in the context of whole-exome sequencing data.

RET kinase is aberrantly activated in thyroid cancers and in rare cases of lung and colon cancer, and has been validated as a molecular target in these tumors. Vandetanib was recently approved for the treatment of medullary thyroid cancer. However, vandetanib is ineffective in vitro against RET mutants carrying bulky aminoacids at position 804, the gatekeeper residue, similarly to drug-resistant BCR-ABL mutants in chronic myeloid leukemia. Ponatinib is a multi-target kinase inhibitor that was recently approved for treatment-refractory Philadelphia-positive leukemia. We show here potent inhibition of oncogenic RET by ponatinib, including the drug-insensitive V804M/L mutants. Ponatinib inhibited the growth of RET+ and BCR-ABL+ cells with similar potency, while not affecting RET-negative cells. Both in biochemical and in cellular assays ponatinib compared favorably with known RET inhibitors, such as vandetanib, cabozantinib, sorafenib, sunitinib and motesanib, used as reference compounds. We suggest that ponatinib should be considered for the treatment of RET+ tumors, in particular those expressing vandetanib-resistant V804M/L mutations., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 μm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

BCR/ABL (Breakpoint Cluster Region protein/Abelson tyrosine-protein kinase 1) kinase domain (KD) mutations represent the most frequently described mechanism of resistance to the treatment with tyrosine kinase inhibitors (TKI) in patients with chronic myeloid leukemia (CML). Mutations may impair TKI activity by directly or indirectly impairing the drug binding to the protein. We report the discovery of three new BCR/ABL mutations, L248R, T315V, and F317R identified in two patients with CML (L248R and T315V) and in one patient with Ph+ acute lymphoblastic leukemia (ALL) (F317R). Mutations were screened against second-generation (bosutinib, nilotinib, and dasatinib), as well as third-generation TKIs (ponatinib/AP-24534 and DCC-2036). Furthermore, the activity profile of ponatinib and DCC-2036 against a panel of 24 clinically relevant BCR/ABL mutants is presented and compared to the other TKIs. The IC50 values for each TKI against the mutants and the IC50 increase over wild type BCR/ABL (relative resistance, RR) were calculated to define four resistance levels: sensitive (RR ≤ 2), moderately resistant (2 < RR ≤ 4), resistant (4 < RR ≤ 10), or highly resistant (RR > 10). L248R and T315V showed high resistance to imatinib, bosutinib, dasatinib, and nilotinib, intermediate resistance to ponatinib, but were sensitive to DCC-2036. Interestingly, F317R showed a moderate resistance to imatinib and nilotinib, but is resistant/highly resistant to dasatinib, bosutinib, ponatinib, and DCC-2036. The availability of drugs activity profiles may become a useful tool for clinicians dealing with the treatment of drug-resistant CML patients.

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.

The ERG gene belongs to the ETS family of transcription factors and has been found to be involved in atypical chromosomal rearrangements in several cancers. To gain insight into the oncogenic activity of ERG, we compared the gene expression profile of NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG. We found that all three ERG fusions significantly up-regulate PIM1 expression in the NIH-3T3 cell line. PIM1 is a serine/threonine kinase frequently over-expressed in cancers of haematological and epithelial origin. We show here that tERG expression induces PIM1 in the non-malignant prostate cell line RWPE-1, strengthening the relation between tERG and PIM1 up-regulation in the initial stages of prostate carcinogenesis. Silencing of tERG reversed PIM1 induction. A significant association between ERG and PIM1 expression in clinical prostate carcinoma specimens was found, suggesting that such a mechanism may be relevant in vivo. Chromatin Immunoprecipitation experiments showed that tERG directly binds to PIM1 promoter in the RWPE-1 prostate cell line, suggesting that tERG could be a direct regulator of PIM1 expression. The up-regulation of PIM1 induced by tERG over-expression significantly modified Cyclin B1 levels and increased the percentage of aneuploid cells in the RWPE-1 cell line after taxane-based treatment. Here we provide the first evidence for an ERG-mediated PIM1 up-regulation in prostate cells in vitro and in vivo, suggesting a direct effect of ERG transcriptional activity in the alteration of genetic stability.

Purpose: The purpose of this study was to evaluate the effectiveness of the platform-switching technique to prevent crestal bone loss following the restoration of dental implants., Materials and Methods: This randomized prospective multicenter trial analyzed 60 partially edentulous adults recruited at 12 professional dental centers. Subjects were randomly selected to receive either platform-enlarged or control cylindric implants in three different surgical procedures: conventional nonsubmerged, submerged, and submerged with a reduced abutment. The primary outcome measure was the change in crestal bone level assessed radiographically 12 and 24 months following placement. Nonparametric analysis of variance for repeated measures (the Friedman test) was used to assess the overall significance over time of the differences among implants in changes in crestal bone levels. Comparisons among and between groups of implants were performed by the nonparametric Friedman and Wilcoxon tests, respectively. In all the analyses an alpha = .05 was considered significant., Results: A total of 360 implants were placed (60 for each group). Three control implants failed during the 2nd year following placement. All submerged and 92% of nonsubmerged platform-enlarged implants exhibited no bone loss. Control implants with an abutment as large as the implant platform exhibited more bone loss than their platform-enlarged counterparts (P < .001) or control implants with a reduced abutment (P < .001). Submerged implants with an enlarged platform showed better crestal bone preservation than submerged control implants with a reduced abutment (P = .06)., Conclusions: The findings of the current trial indicated that the use of implants with an enlarged platform can result in better preservation of crestal bone as compared with conventional cylindric implants when a reduced abutment is mounted.

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584)., Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor., Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib., Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

Self-renewal of Bcr-Abl(+) chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated beta-catenin. Although beta-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of beta-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with beta-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate beta-catenin at Y86 and Y654 residues. This Y-phospho beta-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the beta-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated beta-catenin, which presents an increased binding affinity for the Axin/GSK3beta complex. Although Bcr-Abl does not affect GSK3beta autophosphorylation, it prevents, through its effect on beta-catenin Y phosphorylation, Axin/GSK3beta binding to beta-catenin and its subsequent S/T phosphorylation. Silencing of beta-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl(+) CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of beta-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor, c-Kit, and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency, both in cell-free assays and in cells, thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene, but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition, we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416, but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally, SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases.

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay.

Purpose: The aim of this randomized study was to evaluate and compare the long-term success rates of cylindric, screw-type titanium implants with a larger diameter (5.9 mm) that were placed in fresh extraction sockets in association with resorbable bone substitutes or a resorbable membrane., Materials and Methods: Eighty-three partially edentulous adult patients, selected from among those treated in 1997 and 1998 at the San Raffaele Institute in whom 1 or more implants had been placed into fresh posterior mandibular or maxillary sockets, were included in the study. A total of 111 implants were placed, 36 in mandibles and 75 in maxillae. Fifty-six implants were placed in combination with resorbable hydroxyapatite (HA group) and 55 with a resorbable membrane (MR group). Intraoral radiographs and follow-up examinations, including verification of implant stability via the Periotest, were carried out at second-stage surgery 3, 6, 9, and 12 months later; and then annually up to 4 years after placement of the definitive restoration. The radiographic examination was conducted by means of a standardized procedure to verify osseointegration., Results: There was 100% attendance at the follow-up examination after 4 years. At second-stage surgery, which was performed after 4 to 6 months' healing time, none of the implants showed any signs of mobility, peri-implantitis, or bone loss. Two implants failed in the MR group, one at 3 months and one at 9 months after placement; 1 implant failed in the HA group at 4 months after placement. After 4 years, the implant success rate was 97.3% (108 of 111 implants were considered successful). The success rate did not differ significantly between the HA group (98.2%) and the MR group (96.4%)., Discussion: The use of larger-diameter implants served to minimize the anatomic discrepancies that would have evolved when substituting a molar with a standard-diameter implant. According to the accepted criteria for success, the 5-year success rate should be at least 85%; therefore both methods may be considered satisfactory., Conclusion: Implants placed in combination with a resorbable allogeneic material or with a resorbable membrane provided predictable long-term results when restored with a fixed partial denture.