Impaired Hydroxylation of 5-Methylcytosine In TET2 mutated Patients with Myeloid Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1-1
Author(s):  
Anna Jankowska ◽  
Myunggon Ko ◽  
Yun Huang (equal contribution) ◽  
Utz J. Pape ◽  
Hadrian Szpurka ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Genomic Dna ◽  
Conflicts Of Interest ◽  
Methylation Pattern ◽  
Myeloid Malignancies ◽  
Cpg Sites ◽  
Low Levels ◽  
Methylation Patterns ◽  
The Impact

Abstract Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β>0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

145 THE METHYLATION PATTERNS OF POTENTIAL DIFFERENTIALLY METHYLATED REGIONS IN BOVINE Xist, Impact, NDN, AND H19 GENES

2007 ◽  
Vol 19 (1) ◽  
pp. 190
Author(s):  
N. T. D'Cruz ◽  
K. J. Wilson ◽  
M. K. Holland
Keyword(s):  
Genomic Dna ◽  
Bisulfite Sequencing ◽  
Cpg Island ◽  
Methylation Pattern ◽  
Imprinted Genes ◽  
Aberrant Methylation ◽  
Differentially Methylated Regions ◽  
Methylation Patterns ◽  
The Impact

Clinical and laboratory-assisted reproductive techniques such as ICSI have recently been associated with an increased incidence of several syndromes associated with defects in genomic imprinting. Genomically imprinted genes are expressed from only one parental allele and act to regulate growth of the fetus and placenta and brain development/ function. Imprinted genes are controlled by differentially methylated regions (DMRs), whereby one parental allelle (i.e. either maternal or paternal) is epigenetically silenced via methylation. Studies conducted in vitro suggest that culture of embryos and embryo manipulations may perturb the imprinting process. In the current study, the genomic DNA methylation patterns of CpG islands within bovine H19 (27 CpGs analyzed), Impact (36 CpGs), NDN (22 CpGs), and Xist (21 CpGs) were analyzed by bisulfite sequencing. Genomic DNA from a female fibroblast cell line and sperm were chosen for analysis. Potential DMRs for the 4 genes were identified, and semi-nested PCR primers were designed surrounding those regions. Second-round PCR products (2 separate reactions) were mixed, subcloned, and sequenced (n ≥ 10). The fibroblast methylation pattern of the Xist DMR showed consistent methylation in 50% of sequenced clones, with no methylation observed in sperm. The H19 DMR in fibroblast DNA also showed consistent methylation in 25% of sequenced clones, with sperm DNA fully methylated. These results confirm previous studies showing that Xist and H19 are imprinted in cattle. Sequencing of the putative Impact DMR clones indicated no methylation in either cell type, suggesting no imprinting in cattle, tissue-specific imprinting, or that this CpG island (15 bp post ATG) is not the DMR that controls imprinted expression of the Impact gene. The NDN DMR (500 bp post ATG) in sperm was not methylated, whereas the fibroblast cells had a variable methylation pattern. This may be for the same reasons suggested for Impact, but the variability within the CpG island may also be due to in vitro culture conditions resulting in aberrant methylation. This possible culture effect is currently being confirmed through bisulfite sequencing of the gene in an adult tissue. The investigation of methylation patterns in oocytes is also underway. Together, the information gathered will be used to determine the imprinting status of several bovine genes and, in the future, whether any of these imprinted genes are responsible for the increased pregnancy loss and calf abnormalities associated with advanced reproductive technologies.

The role of implicit learning in logo substitution

2019 ◽  
Vol 36 (5) ◽  
pp. 610-619 ◽  
Author(s):  
Rafal Ohme ◽  
Christo Boshoff
Keyword(s):  
Implicit Learning ◽  
Experimental Studies ◽  
Target Market ◽  
Human Beings ◽  
Content Type ◽  
Quasi Experimental ◽  
Low Levels ◽  
The Impact ◽  
A Company

Purpose Some marketers have challenged psychologists’ contention that human beings can only learn by using conscious effort. They argue that advertising can be effective at low levels of (or even no) attention. Also, despite the absence of (or low levels of) consciousness, these subconscious responses can be linked to brands. The purpose of this study is to investigate the impact of implicit learning in the context of logo substitution – an image that may not look like the original logo, and may not even be consciously associated with the original brand or its logo. Design/methodology/approach Data were collected by means of two quasi-experimental studies. Findings The results suggest that, thanks to implicit learning, logo substitution can be effective. Research limitations/implications One limitation was that data were collected from two relatively small convenience samples. Practical implications Logo substitution can be of value when a company faces a situation when advertising is banned or restricted, when the target market is saturated with marketing stimuli (clutter) and when there is a risk that aggressive advertising can lead to psychological reactance. The purpose of logo substitution would then be to unobtrusively activate mental representations closely related to the original logo. Originality/value The central contribution of this study is that it demonstrates how the principles of implicit social cognition, implicit learning and logo substitution can be used by marketers to overcome the undesirable and even adverse advertising circumstances they sometimes face.

The Role of Beta 2 Integrins in Macrophage Migration During Resolution of Inflammation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3600-3600 ◽  
Author(s):  
Yevgeniya Kushchayeva ◽  
Darya Mishchuk ◽  
Tatiana Ugarova
Keyword(s):  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Initial Population ◽  
Blood Monocytes ◽  
Resident Cell ◽  
Low Levels ◽  
Inflammatory Macrophages ◽  
Β2 Integrins

Abstract Abstract 3600 Poster Board III-537 The mobilization of blood monocytes and their differentiation into macrophages during the immune-inflammatory response helps to prepare the tissue for resolution. During the resolution phase of inflammation macrophages do not die locally: some cells emigrate by draining lymphatics whereas some remain at the site of inflammation. The major myelo-monocytic integrin αMβ2 (Mac-1, CD11b/CD18), together with two related integrins αDβ2 (CD11d/CD18) and αXβ2 (CD11c/CD18), mediate critical adhesive reactions of monocyte/macrophages. However, the roles of these adhesion receptors in control of macrophage retention at sites of inflammation and their emigration to lymph nodes are unclear. Using a mouse model of sterile peritonitis induced by thioglycollate injection, we examined the dynamics of macrophage β2 integrins during the resolution phase of inflammation. Macrophages were defined by FACS analyses as a population of cells expressing αMβ2high, αDβ2+ and CD115+. The initial population of resident β2, positive for βDβ2 and negative for αXβ2. The thioglycollate-challenged mice showed a ∼4-fold increase in macrophages on day 3 followed by a progressive decrease to normal resident cell numbers by day 13. Expression of αMβ2 on macrophages on day 3 decreased by 2.5-fold as a result of dilution of the initial population of αMβ2high resident macrophages by infiltrating blood monocytes expressing αMβ2low. However, after day 3, the density of αMβ2 on macrophages gradually increased and by day 13 returned to the high levels characteristic of resident macrophages. By contrast, expression of αDβ2 and αXβ2 on inflammatory macrophages increased by 2-fold by day 6-9 compared to that on resident macrophages and then returned to the resident levels by day 3. Thus, although the number of macrophages decreased from day 3 to day 9 by several fold, the population of macrophages which remained in the peritoneum was enriched in cells expressing the high levels of αMβ2 and αDα2. Tracking migration of fluorescently labeled peritoneal cells demonstrated that a population of macrophages which leaves the inflamed peritoneum and enters lymph nodes consists of cells expressing low levels of αMβ2 and αDβ2. These data suggested that upregulation of β2 integrins, especially αMβ2, may be responsible for the retention of macrophages in the peritoneum. Indeed, the rate of macrophage emigration from the peritoneum in the αMβ2-deficient mice was significantly higher than that in wild-type mice. The results indicate that macrophage emigration from the inflamed site is controlled by the level of integrin αMβ2 and αDβ2 with low expressors being migratory and high expressors remaining in the peritoneum. The data also highlight the importance of integrins αDβ2 and αXβ2 as specific markers of inflammatory macrophages. Disclosures: No relevant conflicts of interest to declare.

Regulation of Hematopoietic Stem Cell Mitochondrial Metabolism

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-33-SCI-33
Author(s):  
Saghi Ghaffari
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Conflicts Of Interest ◽  
Mitochondrial Metabolism ◽  
Metabolic Switch ◽  
Hematopoietic Stem ◽  
Highly Active ◽  
Approaches To Study ◽  
Low Levels ◽  
The Impact

Abstract Hematopoietic stem cells (HSCs) like most, if not all, adult stem cells are primarily quiescent but have the potential to become highly active on demand. HSC quiescence is maintained by glycolytic metabolism and low levels of reactive oxygen species (ROS), which indicate that mitochondria are relatively inactive in quiescent HSC. However, HSC cycling - and exit of quiescence state - require a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. To improve our understanding of mechanisms that integrate energy metabolism with HSC homeostasis, my laboratory has been focused on the transcription factor FOXO3, which is critical for the maintenance of HSC quiescence and redox state and is implicated in HSC aging. We showed recently that FOXO3 is key to HSC mitochondrial metabolism, independent of its inhibition of ROS or mTOR signaling. Mitochondria divide and fuse constantly in part to segregate and dispose of their damaged counterparts. These processes are influenced by and highly linked to mitochondrial metabolism. We have recently developed imaging approaches to study HSC mitochondrial divisions. Mechanisms by which FOXO3 regulates HSC mitochondria and the impact of impaired FOXO3 on the HSC health and activity, and mitochondrial network will be discussed. Detailed understanding of the mitochondrial metabolism and divisions in HSC and their relationship to nuclear transcription are likely to have broad implications for the state of HSC fitness, regenerative capacity and aging. Disclosures No relevant conflicts of interest to declare.

High mRNA expression of LMO4, a BRCA1 downregulator, correlates with better prognosis in erlotinib-treated non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18137-e18137
Author(s):  
Santiago Viteri Ramirez ◽  
Carlota Costa ◽  
Ana Gimenez Capitan ◽  
Susana Benlloch ◽  
Miquel Taron ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Stage Iv ◽  
Progression Free Survival ◽  
Negative Regulator ◽  
Egfr Mutations ◽  
Mrna Levels ◽  
Breast Cancers ◽  
T790m Mutation ◽  
Low Levels ◽  
The Impact

e18137 Background: Advanced NSCLC p with EGFR activating mutations show an impressive progression-free survival (PFS) to erlotinib. The co-existence of the EGFR T790M mutation, in conjunction with high BRCA1 mRNA levels, affected PFS to erlotinib (Rosell et al. CCR 2011). LMO4 is a negative regulator of BRCA1 function in sporadic breast cancers, and CtIP can bind to BRCA1 and LMO4. We have assessed the expression of CtIP, LMO4 and BRCA1 and examined the impact of CtIP and LMO4 levels on outcome. Methods: mRNA expression of LMO4 and CtIP was examined by RT-PCR in the original pretreatment tumor biopsies of 81 NSCLC p with sensitive EGFR mutations. Results: Expression of BRCA1 and LMO4 was successfully assessed in 55 p: median age, 68; 61.8% female; 98.2% Caucasian; 63.6% never-smokers; 81.8% ECOG PS <2; 80% adenocarcinoma; 14.5% BAC; 4.5% LCC; 94.5% stage IV; 63.6% exon 19 deletion; 36.4% L858R mutation; 36.4% T790M; 83.1% showed clinical benefit to erlotinib (CR/PR/SD). BRCA1 expression was correlated with that of CtIP (r=0.31; P=0.01) and LMO4 (r=0.32; P=0.02). There was no correlation between CtIP and LMO4 (r=0.09; P=0.49). PFS for p with high LMO4 levels was not reached while it was 13 months (m) for p with low levels (P=0.006). Overall survival (OS) was not reached for p with high levels of LMO4 and was 31 m for p with low levels (P=0.17). No differences in PFS or OS were observed according to CtIP levels. When BRCA1 and LMO4 expression was analyzed together, PFS was not reached for p with low BRCA1 and high LMO4 levels and was 19 m for p with low levels of both genes (P=0.04). PFS was 8 m for p with high BRCA1 and low LMO4 levels and 18 m for p with high levels of both genes (P=0.03). In the multivariate analysis, BRCA1 and LMO4 expression emerged as markers of PFS (Table). Conclusions: BRCA1 and LMO4 mRNA expression can predict PFS to erlotinib in p with EGFR mutations and could be useful in the development of new therapeutic strategies. [Table: see text]

scholarly journals Quantitative High-Resolution CpG Island Mapping with Pyrosequencing™ Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia

2007 ◽  
Vol 53 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Kai Brakensiek ◽  
Luzie U Wingen ◽  
Florian Länger ◽  
Hans Kreipe ◽  
Ulrich Lehmann
Keyword(s):  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Cpg Island ◽  
Methylation Status ◽  
Low Grade ◽  
Myeloid Malignancies ◽  
Cpg Sites ◽  
Methylation Patterns ◽  
Cdkn2b Gene ◽  
Disease Specific

Abstract Background: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. Methods: We used Pyrosequencing™ technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. Results: The use of optimized sequencing primers and the new Pyro Q-CpG™ software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over ∼100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. Conclusion: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

scholarly journals Correlation of Lymphoma Patient Information Level with Healthcare Experience

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4782-4782
Author(s):  
Natalie Dren ◽  
Lorna Warwick ◽  
Karen Van Rassel ◽  
Theodoros Moysiadis ◽  
Christina Karamanidou ◽  
...  
Keyword(s):  
Side Effects ◽  
Patient Experience ◽  
Patient Information ◽  
Conflicts Of Interest ◽  
Information Support ◽  
Information Level ◽  
Adequate Information ◽  
Inadequate Information ◽  
Low Levels ◽  
The Impact

Abstract Background: Across recent health reform research, there is growing advocacy and awareness surrounding the idea that patients should act as more effective managers of their health and healthcare. Knowledge dissemination is frequently named as a preliminary requirement for this shift in attitude and behaviors. In 2017, the Lymphoma Coalition (LC) conducted a mixed methods investigation to determine if evidence exists pointing to better outcomes for more 'informed' patients. Though outcome measurements and definitions varied throughout the literature, one theme remained consistent: when a patient has knowledge surrounding their condition, they are more inclined to be confident in sustaining an active patient role, they ask more questions and their patient experience is improved. To continue this investigation, the LC utilized the 2018 Global Patient Survey (GPS) on Lymphomas and CLL to further explore patient awareness and understanding, sources and level of information, support from healthcare professionals (HCPs), and the impact this has on the patient experience. Methods: The 2018 LC GPS was hosted on a third-party portal from January 2018 to March 2018. Patient and caregiver versions were prepared and made available in 19 languages. The survey questions focused on the following: patient information and support, fear of relapse, fatigue, living with side effects, and barriers to care. The survey was advertised through the social media of 65+ lymphoma-related patient organisations, Lymphoma Hub, scientific partners, INTERLYMPH, and HCPs. Overall, 6631 participants took part from all over the world. To perform the analysis, the surveys completed by patients and those completed by caregivers were merged. A minimum completion threshold (0.30) was defined in order to eliminate partially completed surveys. Descriptive statistics were performed for all questions of the survey. Associations between factors were examined through cross-tabulations and chi-square tests (significance level set at p=0.05). All statistical analyses were performed with IBM SPSS v21. The results presented are those specific to the 'patient information and support' sub-investigation. Results: When asked what level of information they felt they had overall, 34% of respondents globally felt they had received adequate information, 45% somewhat adequate and 21% inadequate information. The impact of perceived information level was reflected in respondent's understanding of the medical aspects of their lymphoma, diagnosis and care. Respondents with adequate information reported a greater understanding of all topics surrounding diagnosis and care (subtype, treatments, side effect management) following their initial visit to the doctor (Table 1, Figures 1 & 2). Adequately informed respondents were more confident in determining the need for medical care vs. handling a health problem on their own (59%) compared to somewhat (35%) and inadequately (22%) informed respondents. Similar trends were observed across the majority of feeling and understanding categories (Figure 3). Generally, adequately informed respondents reported experiencing low levels of negative feelings (out of control, fearful) 'most days', while inadequately informed respondents reported experiencing low levels of positive feelings (in control, mentally/physically strong) 'most days'. To analyze doctor-patient communication, somewhat and inadequate information levels were grouped as a comparator against adequate information; across all categories, improved communication was reported by those with adequate information (Figure 4). Additionally, the general reporting of physical, medical, and psycho-social side effects was statistically dependent on the information level variable. Conclusion: Having a perceived adequate information level was correlated with more self-reported positive healthcare experiences. Patients with adequate information reported bettered management of their health and healthcare through improved understanding, confidence levels, and communication. Therefore, access to credible timely information is an important aspect to a successful patient experience. These results present implications for both patient outcomes (health behaviors, health status) and burdens to the healthcare system. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epigenetic Scanning of KEAP1 CpG Sites Uncovers New Molecular-Driven Patterns in Lung Adeno and Squamous Cell Carcinomas

Antioxidants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 904 ◽  
Author(s):  
Federico Pio Fabrizio ◽  
Tommaso Mazza ◽  
Stefano Castellana ◽  
Angelo Sparaneo ◽  
Lucia Anna Muscarella
Keyword(s):  
Squamous Cell ◽  
Epigenetic Silencing ◽  
Related Factor ◽  
Aberrant Methylation ◽  
Prognostic Role ◽  
Protein Levels ◽  
Cpg Sites ◽  
Nsclc Patients ◽  
The Impact

Background: The KEAP1/NRF2 (Kelch-like ECH-associated protein 1/nuclear factor erythroid 2–related factor 2) pathway modulates detoxification processes and participates in the resistance of solid tumors to therapy. Scientific evidence about the presence of genetic and epigenetic abnormalities of the KEAP1 gene was firstly reported in non-small-cell lung cancer (NSCLC) and then described in other tumors. At present, the prognostic role of aberrant methylation at cytosine-guanine dinucleotide (CpG) sites of the KEAP1 gene promoter is debated in NSCLC, and its correlation with transcriptional changes and protein levels remains to be defined in large sample cohorts. Methods: We evaluated and compared multiple KEAP1 omics data (methylation, transcript, and protein expression levels) from The Cancer Genome Atlas (TCGA) to explore the role of CpGs located in different portions of KEAP1 and the correlation between methylation, transcription, and protein levels. Data from two subsets of lung adenocarcinoma (LUAD, n = 617) and lung squamous cell carcinoma (LUSC, n = 571) cohorts of NSCLC patients with different disease stages were evaluated. Results: We found that the methylation levels of many KEAP1 CpGs at various promoter and intragenic locations showed a significant inverse correlation with the transcript levels. Interestingly, these results were limited to the KRAS wild-type LUSC and LUAD cohorts, whereas in LUAD the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in the EGFR mutated subpopulation. Conclusions: These results support the idea that the prognostic role of KEAP1 CpG sites warrants more in-depth investigation and that the impact of their changes in methylation levels may differ among specific NSCLC histologies and molecular backgrounds. Moreover, the observed impact of epigenetic silencing on KEAP1 expression in specific KRAS and EGFR settings may suggest a potential role of KEAP1 methylation as a predictive marker for NSCLC patients for whom anti-EGFR treatments are considered.

The PIM Kinase Inhibitor K00135 Sensitizes Primary Acute Lymphoblastic Leukaemia (ALL) Cells to DNA-Damaging Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3238-3238
Author(s):  
David Da Costa ◽  
Victoria J Weston ◽  
Stefan Knapp ◽  
A. Malcolm R Taylor ◽  
Pamela R. Kearns ◽  
...  
Keyword(s):  
Dna Damage ◽  
Myeloid Leukaemia ◽  
Conflicts Of Interest ◽  
Constitutive Expression ◽  
Treatment Resistance ◽  
Tumour Cells ◽  
Leukaemia Virus ◽  
Pim Kinases ◽  
The Impact

Abstract Abstract 3238 Relapsed ALL is the most common cause of death in children, with only 30% being cured despite intensified treatment and stem cell transplantation. We previously showed that a subset of paediatric ALL tumours have a defect in the apoptotic response to DNA damage in vitro which correlates with a poor clinical response in vivo. Proviral integration site for Moloney murine leukaemia virus (PIM) proteins are a family of serine/threonine kinases which mediate prosurvival signals in response to cytokines, growth factors, hypoxia and DNA damage. PIM kinases have been implicated in several haematological malignancies such as chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML), and T-ALL. However, the role of PIM kinases and their contribution to treatment resistance in a large cohort of primary paediatric ALL has not been comprehensively addressed. To address the frequency of PIM overexpression in ALL we used quantitative real-time PCR to quantify the levels of PIM-1 and PIM-2 in an unselected cohort of 86 primary ALL tumours compared to the PIM-expressing lymphoid cell line K562. We observed high PIM-1 levels in only 2/86 (2.3%) ALLs, whereas PIM-2 was highly expressed in 20/86 (23%) primary tumours. PIM-2 overexpression was detected in tumours with a range of chromosome translocations or hyperdiploidy. In representative samples, the level of PIM-2 protein expression correlated with abundance of mRNA transcript. We then investigated the role of PIM overexpression on apoptosis using the highly specific PIM inhibitor, imidazo[1,2-b]pyridazine K00135, to sensitize tumour cells with high PIM expression to ionising radiation (IR)-induced apoptosis. Pretreatment with K00135 led to sensitization of representative primary tumour cells with high PIM-2 expression to 5Grays IR at an half maximal lethal concentration (LC50) of 10μM, and killing was mediated by caspase-dependant apoptosis. Furthermore, consistent with the role of PIM-2 in the regulation of c-Myc protein stability (Zhang Y, Wang Z, Li X, Magnuson NS. 2008; Oncogene; 27;4890-4819), K00135-induced killing coincided with the downregulation of c-Myc protein. We are currently addressing the impact of PIM inhibition in an ALL xenograft model. Constitutive expression of PIM-2 may be an underlying mechanism of treatment resistance in a subset of paediatric ALL. Pharmacological inhibition of PIM-2 may prove to be a useful therapeutic option for poorly responding ALL in children in the future. Disclosures: No relevant conflicts of interest to declare.

TET2: Mechanism and Functional Consequences of Hydroxymethylation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-32-SCI-32
Author(s):  
Anjana Rao ◽  
Myunggon Ko ◽  
William Pastor ◽  
Yun Huang
Keyword(s):  
Bone Marrow ◽  
Genomic Dna ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Healthy Controls ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Myeloid Neoplasms ◽  
Functional Consequences ◽  
Low Levels

Abstract Abstract SCI-32 TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Somatic TET2 mutations are frequently observed in myeloid neoplasms in humans. Bone marrow samples from patients with mutant TET2, as well as some patients with wild type TET2, display low levels of 5hmC in genomic DNA compared to healthy controls. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool to tailor therapies and assess responses to anticancer drugs. We have developed novel and specific approaches to profile the genomic localization of 5hmC and will describe their application to profiling 5hmC in mouse hematopoietic progenitor cells that express or lack Tet2. Disclosures: No relevant conflicts of interest to declare.

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