Ingvild Haaland

Synergistic induction of p53 mediated apoptosis by valproic acid and nutlin-3 in acute myeloid leukemia

Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rγnull xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.

Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia

The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms β and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.

Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables.

BackgroundTwo-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance.ResultsWe validated this technique on a set of simulated 2DE images, which revealed also correct working under the presence of normalization factors. This was followed by an analysis of p53 2DE immunoblots from cancer cells, known to have unique signaling networks. Since p53 is altered through these signaling networks, we expected to find correlations between the cancer type (acute lymphoblastic leukemia and acute myeloid leukemia) and the p53 profiles. A second correlation analysis revealed a more complex relation between the differentiation stage in acute myeloid leukemia and p53 protein isoforms.ConclusionThe presented analysis method measures relations between 2DE images and external variables without requiring spot detection, thereby enabling the exploration of biosignatures of complex signaling networks in biological systems.

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

BackgroundThe small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.ResultsNutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.ConclusionsAltogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

Proteomics of p53 in diagnostics and therapy of acute myeloid leukemia.

The anti-oncogene TP53 is frequently mutated in human cancer, but in hematological malignancies this is a rare feature. In acute myeloid leukemia (AML) more than 90% of the patients comprise wild type TP53 in their cancer cells, but if TP53 is mutated or deleted the disease is often found to be chemoresistant. In this review we define proteomics of the oncogene product p53 as the study of proteins in the p53 regulating signaling networks, as well as the protein study of members of the p53 family itself. Various messenger RNA splice forms as well as a multitude of post-translational modifications give a high number of protein isoforms in the p53 family. Some of the proteomic techniques allow detection of various isoforms, such as two-dimensional gel electrophoresis in combination with tandem mass spectrometry (MS/MS) and this methodology may therefore increasingly be used as a diagnostic tool in human disease. We introduce the p53 protein as an illustration of the complexity of post-translational modifications that may affect one highly connected protein and discuss the possible impact in AML diagnostics if the p53 profile is reflecting cell stress and status of signal transduction systems of the malignancy.

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML.Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis.ResultsWithin one hour after exposure to ionizing radiation (IR), the AML cells (NB4, MV4-11, HL-60, primary AML cells) showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR) induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis.ConclusionBoth IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s) modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.

cAMP signalling inhibits p53 acetylation and apoptosis via HDAC and SIRT deacetylases

Activation of cAMP signalling potently inhibits DNA damage-induced apoptosis in acute lymphoblastic leukemia cells by promoting the turnover of p53 protein. Recently, we showed that the cAMP-induced destabilization of p53 in DNA-damaged cells occurs as a result of enhanced interaction between p53 and HDM2. In this report, we present results showing that increased levels of cAMP in cells with DNA damage enhances the deacetylation of p53, an event that facilitates the interaction of p53 with HDM2, thus annulling the stabilizing effect of DNA damage on p53. The combined inhibition of the HDAC and SIRT1 deacetylases abolished the cAMP-mediated deacetylation of p53, implying that cAMP-mediated deacetylation of p53 is dependent on the activity of these two classes of histone deacetylases. Importantly, diminishing the activity of HDACs and SIRT1 was also found to reverse the inhibitory effect of cAMP on the DNA damage-induced p53 stabilization and apoptosis, suggesting the involvement of the p53 acetylation pathway in the anti-apoptotic effect of cAMP signalling.

Clinical proteomics of myeloid leukemia

Myeloid leukemias are a heterogeneous group of diseases originating from bone marrow myeloid progenitor cells. Patients with myeloid leukemias can achieve long-term survival through targeted therapy, cure after intensive chemotherapy or short-term survival because of highly chemoresistant disease. Therefore, despite the development of advanced molecular diagnostics, there is an unmet need for efficient therapy that reflects the advanced diagnostics. Although the molecular design of therapeutic agents is aimed at interacting with specific proteins identified through molecular diagnostics, the majority of therapeutic agents act on multiple protein targets. Ongoing studies on the leukemic cell proteome will probably identify a large number of new biomarkers, and the prediction of response to therapy through these markers is an interesting avenue for future personalized medicine. Mass spectrometric protein detection is a fundamental technique in clinical proteomics, and selected tools are presented, including stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ) and multiple reaction monitoring (MRM), as well as single cell determination. We suggest that protein analysis will play not only a supplementary, but also a prominent role in future molecular diagnostics, and we outline how accurate knowledge of the molecular therapeutic targets can be used to monitor therapy response.

The Bergen COPD microbiome study (MicroCOPD): rationale, design, and initial experiences.

Background Recent methodological developments, in particular new sequencing methods for bacterial RNA/DNA, have shown that microorganisms reside in airways that do not suffer from acute infection and that respiratory microbiota might vary according to airways disease status. We aim to establish high-quality sampling methods for lower airways microbiota as well as describe the respiratory microbiome in subjects with and without chronic obstructive pulmonary disease (COPD) and to relate the microbiome to disease development, progression, and the host immune system. Methods The Bergen COPD microbiome study (MicroCOPD) is a longitudinal study aiming to collect data from 200 subjects with COPD as well as 150 individuals without COPD. At baseline, subjects go through a bronchoscopy in which protected specimen brushes, small-volume lavage, bronchoalveolar lavage, and bronchial biopsies provide a unique chance to analyze the microbiota and the host immune system status. These variables will be related to baseline clinical parameters (lung function, smoking status, exacerbation frequency, arterial blood gases, comorbidities, and medications) as well as follow-up parameters (lung function changes, exacerbation frequency, mortality, and more). Results Per date more than 150 bronchoscopies have been performed, equally distributed between cases and controls, with a very low complication frequency. Conclusions MicroCOPD will provide unique data on a large material, with insight on a new field of respiratory research.

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem. Protected bronchoscopic sampling is most suitable for identification of a distinct airway microbiome http://ow.ly/qIIy30eqB9M